Stereochemically enriched compositions for delivery of nucleic acids

ABSTRACT

Provided, in part, is a composition comprising one or more chemical entities of formula I, each of which is a compound of formula I: 
     
       
         
         
             
             
         
       
     
     a pharmaceutically acceptable salt thereof, a solvate thereof, or a solvate of a pharmaceutically acceptable salt thereof, the composition characterized in that greater than a first threshold amount of the total amount of chemical entities of formula I in the composition: are chemical entities of formula I.a, wherein the first threshold amount is 50%; or are chemical entities of formula I.b.1, wherein the first threshold amount is 25%; or are chemical entities of formula I.b.2, wherein the first threshold amount is 25%, wherein the chemical entities of formula I.a, I.b.1, and I.b.2, are described herein, and methods of using such compositions, for example, for the delivery of a polynucleotide in vivo.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/178,142, filed Nov. 1, 2018, which is a continuation of U.S.application Ser. No. 14/749,027, filed Jun. 24, 2015 and issued as U.S.Pat. No. 10,138,213 on Nov. 27, 2018, which claims the benefit of U.S.Provisional Application No. 62/016,512, filed on Jun. 24, 2014, thedisclosures of which are incorporated herein by reference in theirentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 22, 2021, isnamed MRT_1154US3_Sequence_Listing.txt and is 5,563 bytes in size.

BACKGROUND

Delivery of nucleic acids using lipids has been explored extensively fortreatment of various diseases. There remains a great need for lipidsand/or lipids compositions that can deliver nucleic acids, such as shortinterfering RNA (siRNA) and messenger RNA (mRNA) with high efficiencyand low toxicity.

SUMMARY

Among other things, the present invention provides compositionscomprising stereochemically enriched lipids for delivering mRNA. Theinvention is based, in part, on the surprising discovery thatcompositions comprising stereochemically enriched lipid of formula I,below, are highly effective and have unexpectedly low toxicity indelivering mRNA and producing encoded protein in vivo:

The present inventors found that when used for mRNA delivery,stereochemically enriched compositions of I have surprisingly lowtoxicity compared to stereochemically non-enriched, or stereochemicallyless enriched, compositions of the same lipid, as evidenced, forexample, by the dramatically lower alanine aminotransferase (ALT) andaspartate aminotransferase (AST) expression levels. See Table 1.

In some embodiments, the present invention provides a compositioncomprising one or more chemical entities of formula I, each of which isa compound of formula I:

-   a pharmaceutically acceptable salt thereof, a solvate thereof, or a    solvate of a pharmaceutically acceptable salt thereof,-   the composition characterized in that greater than or equal to a    first threshold amount of the total amount of chemical entities of    formula I in the composition are chemical entities of formula I.a,    I.b.1, I.b.2, I.c, I.d, I.e, I.f, I.g, or I.h, each of which is    independently as defined and described below.

In some embodiments, the present invention provides a compositioncomprising one or more chemical entities of formula I, each of which isa compound of formula I:

-   a pharmaceutically acceptable salt thereof, a solvate thereof, or a    solvate of a pharmaceutically acceptable salt thereof,-   the composition characterized in that greater than or equal to a    first threshold amount of the total amount of chemical entities of    formula I in the composition:-   are chemical entities of formula I.a:

-   -   wherein the first threshold amount is 50%; or

-   are chemical entities of formula I.b.1:

-   -   wherein the first threshold amount is 25%; or

-   are chemical entities of formula I.b.2:

-   -   wherein the first threshold amount is 25%.

In some embodiments, the present invention provides a compositioncomprising one or more chemical entities of formula I, each of which isa compound of formula I:

-   a pharmaceutically acceptable salt thereof, a solvate thereof, or a    solvate of a pharmaceutically acceptable salt thereof,-   the composition characterized in that greater than a first threshold    amount of the total amount of chemical entities of formula I in the    composition:-   are chemical entities of formula I.c:

-   -   wherein the first threshold amount is 6.25%; or

-   are chemical entities of formula I.d:

-   -   wherein the first threshold amount is 6.25%; or

-   are chemical entities of formula I.e:

-   -   wherein the first threshold amount is 25%; or

-   are chemical entities of formula I.f:

-   -   wherein the first threshold amount is 25%; or

-   are chemical entities of formula I.g:

-   -   wherein the first threshold amount is 12.5%; or

-   are chemical entities of formula I.h:

-   -   wherein the first threshold amount is 25%.

In some embodiments, the first threshold amount is 50%, 60%, 70%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In someembodiments, the first threshold amount is 50%. In some embodiments, thefirst threshold amount is 70%. In some embodiments, the first thresholdamount is 80%. In some embodiments, the first threshold amount is 95%.

In some embodiments, the present invention provides a compositioncomprising one or more chemical entities of formula I, each of which isa compound of formula I:

-   a pharmaceutically acceptable salt thereof, a solvate thereof, or a    solvate of a pharmaceutically acceptable salt thereof,-   the composition characterized in that greater than or equal to a    first threshold amount of the total amount of chemical entities of    formula I in the composition are chemical entities of a first    formula selected from formulae I.a, I.b.1, I.b.2, I.c, I.d, I.e,    I.f, I.g, or I.h; and-   greater than or equal to a second threshold amount of the total    amount of the chemical entities of the first formula in the    composition are chemical entities of the same stereoisomer of the    first formula.

In some embodiments, greater than or equal to the first threshold amountof the total amount of chemical entities of formula I in the compositionare chemical entities of formula I.a.

In some embodiments, greater than or equal to a second threshold amountof the total amount of chemical entities of formula I.a in thecomposition is a chemical entity of formula I.a.i:

In some embodiments, greater than or equal to a second threshold amountof the total amount of chemical entities of formula I.a in thecomposition is a chemical entity of formula I.a.ii:

In some embodiments, greater than or equal to the first threshold amountof the total amount of chemical entities of formula I in the compositionare chemical entities of formula I.b.1.

In some embodiments, greater than or equal to a second threshold amountof the total amount of chemical entities of formula I.b.1 in thecomposition is a chemical entity of formula I.b.1.i:

In some embodiments, greater than or equal to a second threshold amountof the total amount of chemical entities of formula I.b.1 in thecomposition is a chemical entity of formula I.b.1.ii:

In some embodiments, greater than or equal to the first threshold amountof the total amount of chemical entities of formula I in the compositionare chemical entities of formula I.b.2.

In some embodiments, greater than or equal to a second threshold amountof the total amount of chemical entities of formula I.b.2 in thecomposition is a chemical entity of formula I.b.2.i:

In some embodiments, greater than or equal to a second threshold amountof the total amount of chemical entities of formula I.b.2 in thecomposition is a chemical entity of formula I.b.2.ii:

In some embodiments, the second threshold amount is 50%, 60%, 70%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In someembodiments, the second threshold amount is 50%. In some embodiments,the second threshold amount is 70%. In some embodiments, the secondthreshold amount is 80%. In some embodiments, the second thresholdamount is 95%.

In some embodiments, the present invention provides a compositioncomprising one or more chemical entities of formula I, each of which isa compound of formula I:

-   a pharmaceutically acceptable salt thereof, a solvate thereof, or a    solvate of a pharmaceutically acceptable salt thereof,-   the composition characterized in that greater than or equal to a    third threshold amount of the total amount of chemical entities of    formula I in the composition are chemical entities of formula I.

In some embodiments, the present invention provides a compositioncomprising one or more chemical entities of formula I, each of which isa compound of formula I:

-   a pharmaceutically acceptable salt thereof, a solvate thereof, or a    solvate of a pharmaceutically acceptable salt thereof,-   the composition characterized in that greater than or equal to a    third threshold amount of the total amount of chemical entities of    formula I in the composition are chemical entities of the same    stereoisomer of formula I.

In some embodiments, a stereoisomer of formula I has the structure offormula I.a.i, I.a.ii, I.b.1.i, I.b.1.ii, I.b.2.i, or I.b.2.ii.

In some embodiments, greater than or equal to the third threshold amountof the total amount of the composition is a chemical entity of formulaI.a.i, I.a.ii, I.b.1.i, I.b.1.ii, I.b.2.i, or I.b.2.ii. In someembodiments, greater than or equal to the third threshold amount of thetotal amount of the composition is a chemical entity of formula I.a.i.In some embodiments, greater than or equal to the third threshold amountof the total amount of the composition is a chemical entity of formulaI.a.ii. In some embodiments, greater than or equal to the thirdthreshold amount of the total amount of the composition is a chemicalentity of formula I.b.1.i. In some embodiments, greater than or equal tothe third threshold amount of the total amount of the composition is achemical entity of formula I.b.1.ii. In some embodiments, greater thanor equal to the third threshold amount of the total amount of thecomposition is a chemical entity of formula I.b.2.i. In someembodiments, greater than or equal to the third threshold amount of thetotal amount of the composition is a chemical entity of formulaI.b.2.ii.

In some embodiments, the third threshold amount is 50%, 60%, 70%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% (w/w). In someembodiments, the third threshold amount is 50% (w/w). In someembodiments, the third threshold amount is 70% (w/w). In someembodiments, the third threshold amount is 80% (w/w). In someembodiments, the third threshold amount is 85% (w/w). In someembodiments, the third threshold amount is 95% (w/w).

In some embodiments, a provided composition further comprises one ormore mRNA for mRNA delivery and expression of the encoded protein invivo.

In some embodiments, the present invention provides methods for highlyefficient delivery and expression of mRNA and encoded protein in vivo.In some embodiments, the present invention provides a method of deliveryof mRNA in vivo, comprising administering to a subject in need ofdelivery a provided composition which comprises an mRNA. In someembodiments, due to their low toxicity, a provide composition permitsmore delivered mRNA, higher protein expression level and/or loweradministration frequency, thereby providing a more potent, safer, andmore patent-friendly mRNA therapy.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 depicts the results of in vivo human ASS1 protein production inwild type mouse liver upon treatment with lipid nanoparticles thatinclude compounds of formula I.

DEFINITIONS

In order for the present invention to be more readily understood,certain terms are first defined below. Additional definitions for thefollowing terms and other terms are set forth throughout thespecification. The publications and other reference materials referencedherein to describe the background of the invention and to provideadditional detail regarding its practice are hereby incorporated byreference.

Amino acid: As used herein, term “amino acid,” in its broadest sense,refers to any compound and/or substance that can be incorporated into apolypeptide chain. In some embodiments, an amino acid has the generalstructure H_(E)N—C(H)(R)—COHO. In some embodiments, an amino acid is anaturally occurring amino acid. In some embodiments, an amino acid is asynthetic amino acid; in some embodiments, an amino acid is a d-aminoacid; in some embodiments, an amino acid is an 1-amino acid. “Standardamino acid” refers to any of the twenty standard 1-amino acids commonlyfound in naturally occurring peptides. “Nonstandard amino acid” refersto any amino acid, other than the standard amino acids, regardless ofwhether it is prepared synthetically or obtained from a natural source.As used herein, “synthetic amino acid” encompasses chemically modifiedamino acids, including but not limited to salts, amino acid derivatives(such as amides), and/or substitutions. Amino acids, including carboxyl-and/or amino-terminal amino acids in peptides, can be modified bymethylation, amidation, acetylation, protecting groups, and/orsubstitution with other chemical groups that can change the peptide'scirculating half-life without adversely affecting their activity. Aminoacids may participate in a disulfide bond. Amino acids may comprise oneor posttranslational modifications, such as association with one or morechemical entities (e.g., methyl groups, acetate groups, acetyl groups,phosphate groups, formyl moieties, isoprenoid groups, sulfate groups,polyethylene glycol moieties, lipid moieties, carbohydrate moieties,biotin moieties, etc.). The term “amino acid” is used interchangeablywith “amino acid residue,” and may refer to a free amino acid and/or toan amino acid residue of a peptide. It will be apparent from the contextin which the term is used whether it refers to a free amino acid or aresidue of a peptide.

Animal: As used herein, the term “animal” refers to any member of theanimal kingdom. In some embodiments, “animal” refers to humans, at anystage of development. In some embodiments, “animal” refers to non-humananimals, at any stage of development. In certain embodiments, thenon-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit,a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). Insome embodiments, animals include, but are not limited to, mammals,birds, reptiles, amphibians, fish, insects, and/or worms. In someembodiments, an animal may be a transgenic animal,genetically-engineered animal, and/or a clone.

Approximately or about: As used herein, the term “approximately” or“about,” as applied to one or more values of interest, refers to a valuethat is similar to a stated reference value. In certain embodiments, theterm “approximately” or “about” refers to a range of values that fallwithin 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%,8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greaterthan or less than) of the stated reference value unless otherwise statedor otherwise evident from the context (except where such number wouldexceed 100% of a possible value).

Chemical entity: As used herein, the term “chemical entity” includes acompound, salt, or solvate thereof, or any combination of compounds,salts, or solvates thereof.

Delivery: As used herein, the term “delivery” encompasses both local andsystemic delivery. For example, delivery of mRNA encompasses situationsin which an mRNA is delivered to a target tissue and the encoded proteinis expressed and retained within the target tissue (also referred to as“local distribution” or “local delivery”), and situations in which anmRNA is delivered to a target tissue and the encoded protein isexpressed and secreted into patient's circulation system (e.g., serum)and systematically distributed and taken up by other tissues (alsoreferred to as “systemic distribution” or 37 systemic delivery).

Expression: As used herein, “expression” of a nucleic acid sequencerefers to translation of an mRNA into a polypeptide, assemble multiplepolypeptides (e.g., heavy chain or light chain of antibody) into anintact protein (e.g., antibody) and/or post-translational modificationof a polypeptide or fully assembled protein (e.g., antibody). In thisapplication, the terms “expression” and “production,” and grammaticalequivalent, are used inter-changeably.

Functional: As used herein, a “functional” biological molecule is abiological molecule in a form in which it exhibits a property and/oractivity by which it is characterized.

Half-life: As used herein, the term “half-life” is the time required fora quantity such as nucleic acid or protein concentration or activity tofall to half of its value as measured at the beginning of a time period.

Improve, increase, or reduce: As used herein, the terms “improve,”“increase” or “reduce,” or grammatical equivalents, indicate values thatare relative to a baseline measurement, such as a measurement in thesame individual prior to initiation of the treatment described herein,or a measurement in a control subject (or multiple control subject) inthe absence of the treatment described herein. A “control subject” is asubject afflicted with the same form of disease as the subject beingtreated, who is about the same age as the subject being treated.

In Vitro: As used herein, the term “in vitro” refers to events thatoccur in an artificial environment, e.g., in a test tube or reactionvessel, in cell culture, etc., rather than within a multi-cellularorganism.

In Vivo: As used herein, the term “in vivo” refers to events that occurwithin a multi-cellular organism, such as a human and a non-humananimal. In the context of cell-based systems, the term may be used torefer to events that occur within a living cell (as opposed to, forexample, in vitro systems).

Isolated: As used herein, the term “isolated” refers to a substanceand/or entity that has been (1) separated from at least some of thecomponents with which it was associated when initially produced (whetherin nature and/or in an experimental setting), and/or (2) produced,prepared, and/or manufactured by the hand of man. Isolated substancesand/or entities may be separated from about 10%, about 20%, about 30%,about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98%, about 99%, or more than about 99% of the other componentswith which they were initially associated. In some embodiments, isolatedagents are about 80%, about 85%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,or more than about 99% pure. As used herein, a substance is “pure” if itis substantially free of other components. As used herein, calculationof percent purity of isolated substances and/or entities should notinclude excipients (e.g., buffer, solvent, water, etc.).

Local distribution or delivery: As used herein, the terms “localdistribution,” “local delivery,” or grammatical equivalent, refer totissue specific delivery or distribution. Typically, local distributionor delivery requires a protein (e.g., enzyme) encoded by mRNAs betranslated and expressed intracellularly or with limited secretion thatavoids entering the patient's circulation system.

messenger RNA (mRNA): As used herein, the term “messenger RNA (mRNA)”refers to a polynucleotide that encodes at least one polypeptide. mRNAas used herein encompasses both modified and unmodified RNA. mRNA maycontain one or more coding and non-coding regions. mRNA can be purifiedfrom natural sources, produced using recombinant expression systems andoptionally purified, chemically synthesized, etc. Where appropriate,e.g., in the case of chemically synthesized molecules, mRNA can comprisenucleoside analogs such as analogs having chemically modified bases orsugars, backbone modifications, etc. An mRNA sequence is presented inthe 5′ to 3′ direction unless otherwise indicated. In some embodiments,an mRNA is or comprises natural nucleosides (e.g., adenosine, guanosine,cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine,2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine,5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine,2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine,C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine,2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine,8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine); chemicallymodified bases; biologically modified bases (e.g., methylated bases);intercalated bases; modified sugars (e.g., 2′-fluororibose, ribose,2′-deoxyribose, arabinose, and hexose); and/or modified phosphate groups(e.g., phosphorothioates and 5′-N-phosphoramidite linkages).

In some embodiments, the mRNA comprises one or more nonstandardnucleotide residues. The nonstandard nucleotide residues may include,e.g., 5-methyl-cytidine (“5mC”), pseudouridine, and/or 2-thio-uridine(“2sU”). See, e.g., U.S. Pat. No. 8,278,036 or WO2011012316 for adiscussion of such residues and their incorporation into mRNA. The mRNAmay be RNA, which is defined as RNA in which 25% of U residues are2-thio-uridine and 25% of C residues are 5-methylcytidine. Teachings forthe use of RNA are disclosed US Patent Publication US20120195936 andinternational publication WO2011012316, both of which are herebyincorporated by reference in their entirety. The presence of nonstandardnucleotide residues may render an mRNA more stable and/or lessimmunogenic than a control mRNA with the same sequence but containingonly standard residues. In further embodiments, the mRNA may compriseone or more nonstandard nucleotide residues chosen from isocytosine,pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine,2-aminopurine, inosine, diaminopurine and 2-chloro-6-aminopurinecytosine, as well as combinations of these modifications and othernucleobase modifications. Certain embodiments may further includeadditional modifications to the furanose ring or nucleobase. Additionalmodifications may include, for example, sugar modifications orsubstitutions (e.g., one or more of a 2′-O-alkyl modification, a lockednucleic acid (LNA)). In some embodiments, the RNAs may be complexed orhybridized with additional polynucleotides and/or peptidepolynucleotides (PNA). In embodiments where the sugar modification is a2′-O-alkyl modification, such modification may include, but are notlimited to a 2′-deoxy-2′-fluoro modification, a 2′-O-methylmodification, a 2′-O-methoxyethyl modification and a 2′-deoxymodification. In certain embodiments, any of these modifications may bepresent in 0-100% of the nucleotides—for example, more than 0%, 1%, 10%,25%, 50%, 75%, 85%, 90%, 95%, or 100% of the constituent nucleotidesindividually or in combination.

Nucleic acid: As used herein, the term “nucleic acid,” in its broadestsense, refers to any compound and/or substance that is or can beincorporated into a polynucleotide chain. In some embodiments, a nucleicacid is a compound and/or substance that is or can be incorporated intoa polynucleotide chain via a phosphodiester linkage. In someembodiments, “nucleic acid” refers to individual nucleic acid residues(e.g., nucleotides and/or nucleosides). In some embodiments, “nucleicacid” refers to a polynucleotide chain comprising individual nucleicacid residues. In some embodiments, “nucleic acid” encompasses RNA aswell as single and/or double-stranded DNA and/or cDNA.

Patient: As used herein, the term “patient” or “subject” refers to anyorganism to which a provided composition may be administered, e.g., forexperimental, diagnostic, prophylactic, cosmetic, and/or therapeuticpurposes. Typical patients include animals (e.g., mammals such as mice,rats, rabbits, non-human primates, and/or humans). In some embodiments,a patient is a human. A human includes pre and post natal forms.

Pharmaceutically acceptable: The term “pharmaceutically acceptable” asused herein, refers to substances that, within the scope of soundmedical judgment, are suitable for use in contact with the tissues ofhuman beings and animals without excessive toxicity, irritation,allergic response, or other problem or complication, commensurate with areasonable benefit/risk ratio.

Polymer: As used herein, a “polymer” refers to a compound comprised ofat least 3 (e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,etc.) repeating covalently bound structural units.

Salt: As used herein, the term “salt” or “pharmaceutically acceptablesalt” refers to those salts which are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of humans andlower animals without undue toxicity, irritation, allergic response andthe like, and are commensurate with a reasonable benefit/risk ratio.Pharmaceutically acceptable salts are well known in the art. Forexample, S. M. Berge et al., describes pharmaceutically acceptable saltsin detail in J. Pharmaceutical Sciences (1977) 66:1-19. Pharmaceuticallyacceptable salts of the compounds of this invention include thosederived from suitable inorganic and organic acids and bases. Examples ofpharmaceutically acceptable, nontoxic acid addition salts are salts ofan amino group formed with inorganic acids such as hydrochloric acid,hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid orwith organic acids such as acetic acid, oxalic acid, maleic acid,tartaric acid, citric acid, succinic acid or malonic acid or by usingother methods used in the art such as ion exchange. Otherpharmaceutically acceptable salts include adipate, alginate, ascorbate,aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate,camphorate, camphorsulfonate, citrate, cyclopentanepropionate.digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts,and the like. Salts derived from appropriate bases include alkali metal,alkaline earth metal, ammonium and N+(C1-4alkyl)4 salts. Representativealkali or alkaline earth metal salts include sodium, lithium, potassium,calcium, magnesium, and the like. Further pharmaceutically acceptablesalts include, when appropriate, nontoxic ammonium. quaternary ammonium,and amine cations formed using counterions such as halide, hydroxide,carboxylate, sulfate, phosphate, nitrate, sulfonate and aryl sulfonate.Further pharmaceutically acceptable salts include salts formed from thequarternization of an amine using an appropriate electrophile, e.g., analkyl halide, to form a quarternized alkylated amino salt.

Systemic distribution or delivery: As used herein, the terms “systemicdistribution,” “systemic delivery,” or grammatical equivalent, refer toa delivery or distribution mechanism or approach that affect the entirebody or an entire organism. Typically, systemic distribution or deliveryis accomplished via body's circulation system, e.g., blood stream.Compared to the definition of “local distribution or delivery.”

Subject: As used herein, the term “subject” refers to a human or anynon-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine,sheep, horse or primate). A human includes pre- and post-natal forms. Inmany embodiments, a subject is a human being. A subject can be apatient, which refers to a human presenting to a medical provider fordiagnosis or treatment of a disease. The term “subject” is used hereininterchangeably with “individual” or “patient.” A subject can beafflicted with or is susceptible to a disease or disorder but may or maynot display symptoms of the disease or disorder.

Substantially: As used herein, the term “substantially” refers to thequalitative condition of exhibiting total or near-total extent or degreeof a characteristic or property of interest. One of ordinary skill inthe biological arts will understand that biological and chemicalphenomena rarely, if ever, go to completion and/or proceed tocompleteness or achieve or avoid an absolute result. The term“substantially” is therefore used herein to capture the potential lackof completeness inherent in many biological and chemical phenomena.

Target tissues: As used herein, the term “target tissues” refers to anytissue that is affected by a disease to be treated. In some embodiments,target tissues include those tissues that display disease-associatedpathology, symptom, or feature.

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” of a therapeutic agent means anamount that is sufficient, when administered to a subject suffering fromor susceptible to a disease, disorder, and/or condition, to treat,diagnose, prevent, and/or delay the onset of the symptom(s) of thedisease, disorder, and/or condition. It will be appreciated by those ofordinary skill in the art that a therapeutically effective amount istypically administered via a dosing regimen comprising at least one unitdose.

Treating: As used herein, the term “treat,” “treatment,” or “treating”refers to any method used to partially or completely alleviate,ameliorate, relieve, inhibit, prevent, delay onset of, reduce severityof and/or reduce incidence of one or more symptoms or features of aparticular disease, disorder, and/or condition. Treatment may beadministered to a subject who does not exhibit signs of a disease and/orexhibits only early signs of the disease for the purpose of decreasingthe risk of developing pathology associated with the disease.

DETAILED DESCRIPTION

The present invention provides, among other things, lipid compositionsand methods for delivering mRNA in vivo using stereochemically enrichedlipid compositions.

Lipid Compositions

In some embodiments, the present invention provides a compositioncomprising one or more chemical entities of formula I, each of which isa compound of formula I, a pharmaceutically acceptable salt thereof, asolvate thereof, or a solvate of a pharmaceutically acceptable saltthereof,

the composition characterized in that greater than or equal to a firstthreshold amount of the total amount of chemical entities of formula Iin the composition are chemical entities of formula I.a, I.b.1, I.b.2,I.c, I.d, I.e, I.f, I.g, or I.h.

In some embodiments, a provided composition is characterized in thatgreater than a first threshold amount of the total amount of chemicalentities of formula I in the composition are chemical entities offormula I.a, I.b.1, I.b.2, I.c, I.d, I.e, I.f, I.g, or I.h. In someembodiments, a provided composition is characterized in that a firstthreshold amount of the total amount of chemical entities of formula Iin the composition are chemical entities of formula I.a, I.b.1, I.b.2,I.c, I.d, I.e, I.f, I.g, or I.h.

As used herein, a “chemical entity” of a formula is a compound of theformula, a pharmaceutically acceptable salt thereof, a solvate thereof,or a solvate of a pharmaceutically acceptable salt thereof.

In some embodiments, the first threshold amount is 50%, 60%, 70%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In someembodiments, the first threshold amount is 50%. In some embodiments, thefirst threshold amount is 70%. In some embodiments, the first thresholdamount is 80%. In some embodiments, the first threshold amount is 95%.

In some embodiments, the composition is characterized in that greaterthan or equal to a first threshold amount of the total amount ofchemical entities of formula I in the composition are chemical entitiesof formula I.a. In some embodiments, the composition is characterized inthat greater than a first threshold amount of the total amount ofchemical entities of formula I in the composition are chemical entitiesof formula I.a. In some embodiments, the composition is characterized inthat a first threshold amount of the total amount of chemical entitiesof formula I in the composition are chemical entities of formula I.a. Insome embodiments, a first threshold amount is 50%. In some embodiments,a first threshold amount is 70%. In some embodiments, a first thresholdamount is 80%. In some embodiments, a first threshold amount is 90%. Insome embodiments, a first threshold amount is 95%.

In some embodiments, the composition is characterized in that greaterthan or equal to a first threshold amount of the total amount ofchemical entities of formula I in the composition are chemical entitiesof formula I.b.1. In some embodiments, the composition is characterizedin that greater than a first threshold amount of the total amount ofchemical entities of formula I in the composition are chemical entitiesof formula I.b.1. In some embodiments, the composition is characterizedin that a first threshold amount of the total amount of chemicalentities of formula I in the composition are chemical entities offormula I.b.1. In some embodiments, a first threshold amount is 25%. Insome embodiments, a first threshold amount is 50%. In some embodiments,a first threshold amount is 70%. In some embodiments, a first thresholdamount is 80%. In some embodiments, a first threshold amount is 90%. Insome embodiments, a first threshold amount is 95%.

In some embodiments, the composition is characterized in that greaterthan or equal to a first threshold amount of the total amount ofchemical entities of formula I in the composition are chemical entitiesof formula I.b.2. In some embodiments, the composition is characterizedin that greater than a first threshold amount of the total amount ofchemical entities of formula I in the composition are chemical entitiesof formula I.b.2. In some embodiments, the composition is characterizedin that a first threshold amount of the total amount of chemicalentities of formula I in the composition are chemical entities offormula I.b.2. In some embodiments, a first threshold amount is 25%. Insome embodiments, a first threshold amount is 50%. In some embodiments,a first threshold amount is 70%. In some embodiments, a first thresholdamount is 80%. In some embodiments, a first threshold amount is 90%. Insome embodiments, a first threshold amount is 95%.

In some embodiments, the composition is characterized in that greaterthan or equal to a first threshold amount of the total amount ofchemical entities of formula I in the composition are chemical entitiesof formula I.c. In some embodiments, the composition is characterized inthat greater than a first threshold amount of the total amount ofchemical entities of formula I in the composition are chemical entitiesof formula I.c. In some embodiments, the composition is characterized inthat a first threshold amount of the total amount of chemical entitiesof formula I in the composition are chemical entities of formula I.c. Insome embodiments, a first threshold amount is 6.25%. In someembodiments, a first threshold amount is 12.5%. In some embodiments, afirst threshold amount is 25%. In some embodiments, a first thresholdamount is 50%. In some embodiments, a first threshold amount is 70%. Insome embodiments, a first threshold amount is 80%. In some embodiments,a first threshold amount is 90%. In some embodiments, a first thresholdamount is 95%.

In some embodiments, the composition is characterized in that greaterthan or equal to a first threshold amount of the total amount ofchemical entities of formula I in the composition are chemical entitiesof formula I.d. In some embodiments, the composition is characterized inthat greater than a first threshold amount of the total amount ofchemical entities of formula I in the composition are chemical entitiesof formula I.d. In some embodiments, the composition is characterized inthat a first threshold amount of the total amount of chemical entitiesof formula I in the composition are chemical entities of formula I.d. Insome embodiments, a first threshold amount is 6.25%. In someembodiments, a first threshold amount is 12.5%. In some embodiments, afirst threshold amount is 25%. In some embodiments, a first thresholdamount is 50%. In some embodiments, a first threshold amount is 70%. Insome embodiments, a first threshold amount is 80%. In some embodiments,a first threshold amount is 90%. In some embodiments, a first thresholdamount is 95%.

In some embodiments, the composition is characterized in that greaterthan or equal to a first threshold amount of the total amount ofchemical entities of formula I in the composition are chemical entitiesof formula I.e. In some embodiments, the composition is characterized inthat greater than a first threshold amount of the total amount ofchemical entities of formula I in the composition are chemical entitiesof formula I.e. In some embodiments, the composition is characterized inthat a first threshold amount of the total amount of chemical entitiesof formula I in the composition are chemical entities of formula I.e. Insome embodiments, a first threshold amount is 25%. In some embodiments,a first threshold amount is 50%. In some embodiments, a first thresholdamount is 70%. In some embodiments, a first threshold amount is 80%. Insome embodiments, a first threshold amount is 90%. In some embodiments,a first threshold amount is 95%.

In some embodiments, the composition is characterized in that greaterthan or equal to a first threshold amount of the total amount ofchemical entities of formula I in the composition are chemical entitiesof formula I.f. In some embodiments, the composition is characterized inthat greater than a first threshold amount of the total amount ofchemical entities of formula I in the composition are chemical entitiesof formula I.f. In some embodiments, the composition is characterized inthat a first threshold amount of the total amount of chemical entitiesof formula I in the composition are chemical entities of formula I.f. Insome embodiments, a first threshold amount is 25%. In some embodiments,a first threshold amount is 50%. In some embodiments, a first thresholdamount is 70%. In some embodiments, a first threshold amount is 80%. Insome embodiments, a first threshold amount is 90%. In some embodiments,a first threshold amount is 95%.

In some embodiments, the composition is characterized in that greaterthan or equal to a first threshold amount of the total amount ofchemical entities of formula I in the composition are chemical entitiesof formula I.g. In some embodiments, the composition is characterized inthat greater than a first threshold amount of the total amount ofchemical entities of formula I in the composition are chemical entitiesof formula I.g. In some embodiments, the composition is characterized inthat a first threshold amount of the total amount of chemical entitiesof formula I in the composition are chemical entities of formula I.g. Insome embodiments, a first threshold amount is 12.5%. In someembodiments, a first threshold amount is 25%. In some embodiments, afirst threshold amount is 50%. In some embodiments, a first thresholdamount is 70%. In some embodiments, a first threshold amount is 80%. Insome embodiments, a first threshold amount is 90%. In some embodiments,a first threshold amount is 95%.

In some embodiments, the composition is characterized in that greaterthan or equal to a first threshold amount of the total amount ofchemical entities of formula I in the composition are chemical entitiesof formula I.h. In some embodiments, the composition is characterized inthat greater than a first threshold amount of the total amount ofchemical entities of formula I in the composition are chemical entitiesof formula I.h. In some embodiments, the composition is characterized inthat a first threshold amount of the total amount of chemical entitiesof formula I in the composition are chemical entities of formula I.h. Insome embodiments, a first threshold amount is 25%. In some embodiments,a first threshold amount is 50%. In some embodiments, a first thresholdamount is 70%. In some embodiments, a first threshold amount is 80%. Insome embodiments, a first threshold amount is 90%. In some embodiments,a first threshold amount is 95%.

In some embodiments, the present invention provides a compositioncomprising one or more chemical entities of formula I, each of which isa compound of formula I:

-   a pharmaceutically acceptable salt thereof, a solvate thereof, or a    solvate of a pharmaceutically acceptable salt thereof,-   the composition characterized in that greater than or equal to a    first threshold amount of the total amount of chemical entities of    formula I in the composition are chemical entities of a first    formula selected from formulae I.a, I.b.1, I.b.2, I.c, I.d, I.e,    I.f, I.g, and I.h; and-   greater than or equal to a second threshold amount of the total    amount of the chemical entities of the first formula in the    composition are chemical entities of the same stereoisomer of the    first formula.

In some embodiments, greater than a first threshold amount of the totalamount of chemical entities of formula I in the composition are chemicalentities of a first formula selected from formulae I.a, I.b.1, I.b.2,I.c, I.d, I.e, I.f, I.g, and I.h. In some embodiments, a first thresholdamount of the total amount of chemical entities of formula I in thecomposition are chemical entities of a first formula selected fromformulae I.a, I.b.1, I.b.2, I.c, I.d, I.e, I.f, I.g, and I.h. In someembodiments, greater than a second threshold amount of the total amountof the chemical entities of the first formula in the composition arechemical entities of the same stereoisomer of the first formula. In someembodiments, a second threshold amount of the total amount of thechemical entities of the first formula in the composition are chemicalentities of the same stereoisomer of the first formula.

In some embodiments, a first formula is formula I.a. In someembodiments, greater than or equal to the first threshold amount of thetotal amount of chemical entities of formula I in the composition arechemical entities of formula I.a, and greater than or equal to a secondthreshold amount of the total amount of the chemical entities of formulaI.a in the composition are chemical entities of the same stereoisomer offormula I.a.

In some embodiments, a stereoisomer of formula I.a has the structure offormula I.a.i:

In some embodiments, a stereoisomer of formula I.a has the structure offormula I.a.ii:

In some embodiments, a stereoisomer of formula I.a has the structure offormula I.a.ii:

In some embodiments, a stereoisomer of formula I.a has the structure offormula I.a.iv:

In some embodiments, a stereoisomer of formula I.a has the structure offormula I.a.v:

In some embodiments, a stereoisomer of formula I.a has the structure offormula I.a.vi:

In some embodiments, a stereoisomer of formula I.a has the structure offormula I.a.vii:

In some embodiments, a stereoisomer of formula I.a has the structure offormula I.a.viii:

In some embodiments, a stereoisomer of formula I.a has the structure offormula I.a.ix:

In some embodiments, a stereoisomer of formula I.a has the structure offormula I.a.x:

In some embodiments, greater than or equal to a second threshold amountof the total amount of chemical entities of formula I.a in thecomposition are chemical entities of formula I.a.i. In some embodiments,greater than or equal to a second threshold amount of the total amountof chemical entities of formula I.a in the composition are chemicalentities of formula I.a.ii. In some embodiments, greater than or equalto a second threshold amount of the total amount of chemical entities offormula I.a in the composition are chemical entities of formula I.a.iii.In some embodiments, greater than or equal to a second threshold amountof the total amount of chemical entities of formula I.a in thecomposition are chemical entities of formula I.a.iv. In someembodiments, greater than or equal to a second threshold amount of thetotal amount of chemical entities of formula I.a in the composition arechemical entities of formula I.a.v. In some embodiments, greater than orequal to a second threshold amount of the total amount of chemicalentities of formula I.a in the composition are chemical entities offormula I.a.vi. In some embodiments, greater than or equal to a secondthreshold amount of the total amount of chemical entities of formula I.ain the composition are chemical entities of formula I.a.vii. In someembodiments, greater than or equal to a second threshold amount of thetotal amount of chemical entities of formula I.a in the composition arechemical entities of formula I.a.viii. In some embodiments, greater thanor equal to a second threshold amount of the total amount of chemicalentities of formula I.a in the composition are chemical entities offormula I.a.ix.

In some embodiments, a first formula is formula I.b.1. In someembodiments, greater than or equal to the first threshold amount of thetotal amount of chemical entities of formula I in the composition arechemical entities of formula I.b.1, and greater than or equal to asecond threshold amount of the total amount of the chemical entities offormula I.b.1 in the composition are chemical entities of the samestereoisomer of formula I.b.1.

In some embodiments, a stereoisomer of formula I.b.1 has the structureof formula I.b.1.i (i.e., R4-SS-cKK-E12).

In some embodiments, a stereoisomer of formula I.b.1 has the structureof formula I.b.1.ii (i.e., S4-SS-cKK-E12).

In some embodiments, a stereoisomer of formula I.b.1 has the structureof formula I.b.1.iii:

In some embodiments, a stereoisomer of formula I.b.1 has the structureof formula I.b.1.iv:

In some embodiments, a stereoisomer of formula I.b.1 has the structureof formula I.b.1.v:

In some embodiments, a stereoisomer of formula I.b.1 has the structureof formula I.b.1.vi:

In some embodiments, greater than or equal to a second threshold amountof the total amount of chemical entities of formula I.b.1 in thecomposition are chemical entities of formula I.b.1.i. In someembodiments, greater than or equal to a second threshold amount of thetotal amount of chemical entities of formula I.b.1 in the compositionare chemical entities of formula I.b.1.ii. In some embodiments, greaterthan or equal to a second threshold amount of the total amount ofchemical entities of formula I.b.1 in the composition are chemicalentities of formula I.b.1.iii. In some embodiments, greater than orequal to a second threshold amount of the total amount of chemicalentities of formula I.b.1 in the composition are chemical entities offormula I.b.1.iv. In some embodiments, greater than or equal to a secondthreshold amount of the total amount of chemical entities of formulaI.b.1 in the composition are chemical entities of formula I.b.1.v. Insome embodiments, greater than or equal to a second threshold amount ofthe total amount of chemical entities of formula I.b.1 in thecomposition are chemical entities of formula I.b.1.vi.

In some embodiments, a first formula is formula I.b.2. In someembodiments, greater than or equal to the first threshold amount of thetotal amount of chemical entities of formula I in the composition arechemical entities of formula I.b.2, and greater than or equal to asecond threshold amount of the total amount of the chemical entities offormula I.b.2 in the composition are chemical entities of the samestereoisomer of formula I.b.2.

In some embodiments, a stereoisomer of formula I.b.2 has the structureof formula I.b.2.i (i.e., R4-RR-cKK-E12).

In some embodiments, a stereoisomer of formula I.b.2 has the structureof formula I.b.2.ii (i.e., S4-RR-cKK-E12).

In some embodiments, a stereoisomer of formula I.b.2 has the structureof formula I.b.2.iii:

In some embodiments, a stereoisomer of formula I.b.2 has the structureof formula I.b.2.iv:

In some embodiments, a stereoisomer of formula I.b.2 has the structureof formula I.b.2.v:

In some embodiments, a stereoisomer of formula I.b.2 has the structureof formula I.b.2.vi:

In some embodiments, greater than or equal to a second threshold amountof the total amount of chemical entities of formula I.b.2 in thecomposition are chemical entities of formula I.b.2.i. In someembodiments, greater than or equal to a second threshold amount of thetotal amount of chemical entities of formula I.b.2 in the compositionare chemical entities of formula I.b.2.ii. In some embodiments, greaterthan or equal to a second threshold amount of the total amount ofchemical entities of formula I.b.2 in the composition are chemicalentities of formula I.b.2.iii. In some embodiments, greater than orequal to a second threshold amount of the total amount of chemicalentities of formula I.b.2 in the composition are chemical entities offormula I.b.2.iv. In some embodiments, greater than or equal to a secondthreshold amount of the total amount of chemical entities of formulaI.b.2 in the composition are chemical entities of formula I.b.2.v. Insome embodiments, greater than or equal to a second threshold amount ofthe total amount of chemical entities of formula I.b.2 in thecomposition are chemical entities of formula I.b.2.vi.

In some embodiments, a first formula is formula I.c. In someembodiments, greater than or equal to the first threshold amount of thetotal amount of chemical entities of formula I in the composition arechemical entities of formula I.c, and greater than or equal to a secondthreshold amount of the total amount of the chemical entities of formulaI.c in the composition are chemical entities of the same stereoisomer offormula I.c. In some embodiments, a stereoisomer of formula I.e has thestructure of formula I.a.i. In some embodiments, a stereoisomer offormula I.e has the structure of formula I.b.1.i. In some embodiments, astereoisomer of formula I.e has the structure of formula I.b.2.i. Insome embodiments, greater than or equal to a second threshold amount ofthe total amount of chemical entities of formula I.e in the compositionare chemical entities of formula I.a.i. In some embodiments, greaterthan or equal to a second threshold amount of the total amount ofchemical entities of formula I.e in the composition are chemicalentities of formula I.b.1.i. In some embodiments, greater than or equalto a second threshold amount of the total amount of chemical entities offormula I.c in the composition are chemical entities of formula I.b.2.i.

In some embodiments, a first formula is formula I.d. In someembodiments, greater than or equal to the first threshold amount of thetotal amount of chemical entities of formula I in the composition arechemical entities of formula I.d, and greater than or equal to a secondthreshold amount of the total amount of the chemical entities of formulaI.d in the composition are chemical entities of the same stereoisomer offormula I.d. In some embodiments, a stereoisomer of formula I.d has thestructure of formula I.a.ii. In some embodiments, a stereoisomer offormula I.d has the structure of formula I.b.1.ii. In some embodiments,a stereoisomer of formula I.d has the structure of formula I.b.2.ii. Insome embodiments, greater than or equal to a second threshold amount ofthe total amount of chemical entities of formula I.d in the compositionare chemical entities of formula I.a.ii. In some embodiments, greaterthan or equal to a second threshold amount of the total amount ofchemical entities of formula I.d in the composition are chemicalentities of formula I.b.1.ii. In some embodiments, greater than or equalto a second threshold amount of the total amount of chemical entities offormula I.d in the composition are chemical entities of formulaI.b.2.ii.

In some embodiments, a first formula is formula I.e. In someembodiments, greater than or equal to the first threshold amount of thetotal amount of chemical entities of formula I in the composition arechemical entities of formula I.e, and greater than or equal to a secondthreshold amount of the total amount of the chemical entities of formulaI.e in the composition are chemical entities of the same stereoisomer offormula I.e. In some embodiments, a stereoisomer of formula I.e has thestructure of formula I.a.iii. In some embodiments, a stereoisomer offormula I.e has the structure of formula I.a.v. In some embodiments, astereoisomer of formula I.e has the structure of formula I.b.1.iii. Insome embodiments, a stereoisomer of formula I.e has the structure offormula I.b.2.iii. In some embodiments, greater than or equal to asecond threshold amount of the total amount of chemical entities offormula I.e in the composition are chemical entities of formula I.a.iii.In some embodiments, greater than or equal to a second threshold amountof the total amount of chemical entities of formula I.e in thecomposition are chemical entities of formula I.a.v. In some embodiments,greater than or equal to a second threshold amount of the total amountof chemical entities of formula I.e in the composition are chemicalentities of formula I.b.1.iii. In some embodiments, greater than orequal to a second threshold amount of the total amount of chemicalentities of formula I.e in the composition are chemical entities offormula I.b.2.iii.

In some embodiments, a first formula is formula I.f. In someembodiments, greater than or equal to the first threshold amount of thetotal amount of chemical entities of formula I in the composition arechemical entities of formula I.f, and greater than or equal to a secondthreshold amount of the total amount of the chemical entities of formulaI.f in the composition are chemical entities of the same stereoisomer offormula I.f. In some embodiments, a stereoisomer of formula I.f has thestructure of formula I.a.vii. In some embodiments, a stereoisomer offormula I.f has the structure of formula I.a.ix. In some embodiments, astereoisomer of formula I.f has the structure of formula I.b.1.vi. Insome embodiments, a stereoisomer of formula I.f has the structure offormula I.b.2.vi. In some embodiments, greater than or equal to a secondthreshold amount of the total amount of chemical entities of formula I.fin the composition are chemical entities of formula I.a.iii. In someembodiments, greater than or equal to a second threshold amount of thetotal amount of chemical entities of formula I.f in the composition arechemical entities of formula I.a.ix. In some embodiments, greater thanor equal to a second threshold amount of the total amount of chemicalentities of formula I.f in the composition are chemical entities offormula I.b.1.vi. In some embodiments, greater than or equal to a secondthreshold amount of the total amount of chemical entities of formula I.fin the composition are chemical entities of formula I.b.2.vi.

In some embodiments, a first formula is formula I.g. In someembodiments, greater than or equal to the first threshold amount of thetotal amount of chemical entities of formula I in the composition arechemical entities of formula I.g, and greater than or equal to a secondthreshold amount of the total amount of the chemical entities of formulaI.g in the composition are chemical entities of the same stereoisomer offormula I.g. In some embodiments, a stereoisomer of formula I.g has thestructure of formula I.a.iv. In some embodiments, a stereoisomer offormula I.g has the structure of formula I.a.viii. In some embodiments,a stereoisomer of formula I.g has the structure of formula I.b.1.iv. Insome embodiments, a stereoisomer of formula I.g has the structure offormula I.b.2.iv. In some embodiments, greater than or equal to a secondthreshold amount of the total amount of chemical entities of formula I.gin the composition are chemical entities of formula I.a.iv. In someembodiments, greater than or equal to a second threshold amount of thetotal amount of chemical entities of formula I.g in the composition arechemical entities of formula I.a.viii. In some embodiments, greater thanor equal to a second threshold amount of the total amount of chemicalentities of formula I.g in the composition are chemical entities offormula I.b.1.iv. In some embodiments, greater than or equal to a secondthreshold amount of the total amount of chemical entities of formula I.gin the composition are chemical entities of formula I.b.2.iv.

In some embodiments, a first formula is formula I.h. In someembodiments, greater than or equal to the first threshold amount of thetotal amount of chemical entities of formula I in the composition arechemical entities of formula I.h, and greater than or equal to a secondthreshold amount of the total amount of the chemical entities of formulaI.h in the composition are chemical entities of the same stereoisomer offormula I.h. In some embodiments, a stereoisomer of formula I.h has thestructure of formula I.a.vi. In some embodiments, a stereoisomer offormula I.h has the structure of formula I.b.1.v. In some embodiments, astereoisomer of formula I.h has the structure of formula I.b.2.v. Insome embodiments, greater than or equal to a second threshold amount ofthe total amount of chemical entities of formula I.h in the compositionare chemical entities of formula I.a.vi. In some embodiments, greaterthan or equal to a second threshold amount of the total amount ofchemical entities of formula I.h in the composition are chemicalentities of formula I.b.1.v. In some embodiments, greater than or equalto a second threshold amount of the total amount of chemical entities offormula I.h in the composition are chemical entities of formula I.b.2.v.

In some embodiments, the second threshold amount is 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99%. In some embodiments, the second threshold amount is 50%. In someembodiments, the second threshold amount is 70%. In some embodiments,the second threshold amount is 80%. In some embodiments, the secondthreshold amount is 85%. In some embodiments, the second thresholdamount is 90%. In some embodiments, the second threshold amount is 95%.

In some embodiments, the present invention provides a compositioncomprising one or more chemical entities of formula I, each of which isa compound of formula I:

-   a pharmaceutically acceptable salt thereof, a solvate thereof, or a    solvate of a pharmaceutically acceptable salt thereof,-   the composition characterized in that greater than or equal to a    third threshold amount of the total amount of chemical entities of    formula I in the composition are chemical entities of formula I.

In some embodiments, a provided composition is characterized in thatgreater than a third threshold amount of the total amount of chemicalentities of formula I in the composition are chemical entities offormula I. In some embodiments, a provided composition is characterizedin that a third threshold amount of the total amount of chemicalentities of formula I in the composition are chemical entities offormula I.

In some embodiments, a provided composition is characterized in thatgreater than or equal to a third threshold amount of the total amount ofchemical entities of formula I in the composition are chemical entitiesof formula I.a, I.b.1, I.b.2, I.c, I.d, I.e, I.f, I.g, or I.h. In someembodiments, a provided composition is characterized in that greaterthan or equal to a third threshold amount of the total amount ofchemical entities of formula I in the composition are chemical entitiesof formula I.a. In some embodiments, a provided composition ischaracterized in that greater than or equal to a third threshold amountof the total amount of chemical entities of formula I in the compositionare chemical entities of formula I.b.1. In some embodiments, a providedcomposition is characterized in that greater than or equal to a thirdthreshold amount of the total amount of chemical entities of formula Iin the composition are chemical entities of formula I.b.2. In someembodiments, a provided composition is characterized in that greaterthan or equal to a third threshold amount of the total amount ofchemical entities of formula I in the composition are chemical entitiesof formula I.c. In some embodiments, a provided composition ischaracterized in that greater than or equal to a third threshold amountof the total amount of chemical entities of formula I in the compositionare chemical entities of formula I.d. In some embodiments, a providedcomposition is characterized in that greater than or equal to a thirdthreshold amount of the total amount of chemical entities of formula Iin the composition are chemical entities of formula I.e. In someembodiments, a provided composition is characterized in that greaterthan or equal to a third threshold amount of the total amount ofchemical entities of formula I in the composition are chemical entitiesof formula I.f. In some embodiments, a provided composition ischaracterized in that greater than or equal to a third threshold amountof the total amount of chemical entities of formula I in the compositionare chemical entities of formula I.g. In some embodiments, a providedcomposition is characterized in that greater than or equal to a thirdthreshold amount of the total amount of chemical entities of formula Iin the composition are chemical entities of formula I.h.

In some embodiments, a provided composition is characterized in thatgreater than or equal to a third threshold amount of the total amount ofchemical entities of formula I in the composition are chemical entitiesof formula I.a.i, I.a.ii, I.a.iii, I.a.iv, I.a.v, I.a.vi, I.a.vii,I.a.viii, I.a.ix, I.b.1.i, I.b.1.ii, I.b.1.iii, I.b.1.iv, I.b.1.v,I.b.1.vi, I.b.2.i, I.b.2.ii, I.b.2.iii, I.b.2.iv, I.b.2.v, and I.b.2.vi.

In some embodiments, the present invention provides a compositioncomprising one or more chemical entities of formula I, each of which isa compound of formula I:

-   a pharmaceutically acceptable salt thereof, a solvate thereof, or a    solvate of a pharmaceutically acceptable salt thereof,-   the composition characterized in that greater than or equal to a    third threshold amount of the total amount of chemical entities of    formula I in the composition are chemical entities of the same    stereoisomer of formula I.

In some embodiments, a provided composition is characterized in thatgreater than a third threshold amount of the total amount of chemicalentities of formula I in the composition are chemical entities of thesame stereoisomer of formula I. In some embodiments, a providedcomposition is characterized in that a third threshold amount of thetotal amount of chemical entities of formula I in the composition arechemical entities of the same stereoisomer of formula I.

In some embodiments, a stereoisomer of formula I has the structure offormula I.a.i, I.a.ii, I.a.iii, I.a.iv, I.a.v, I.a.vi, I.a.vii,I.a.viii, or I.a.ix. In some embodiments, a stereoisomer of formula Ihas the structure of formula I.a.i. In some embodiments, a stereoisomerof formula I has the structure of formula I.a.ii. In some embodiments, astereoisomer of formula I has the structure of formula I.a.iii. In someembodiments, a stereoisomer of formula I has the structure of formulaI.a.iv. In some embodiments, a stereoisomer of formula I has thestructure of formula I.a.v. In some embodiments, a stereoisomer offormula I has the structure of formula I.a.vi. In some embodiments, astereoisomer of formula I has the structure of formula I.a.vii. In someembodiments, a stereoisomer of formula I has the structure of formulaI.a.viii. In some embodiments, a stereoisomer of formula I has thestructure of formula I.a.ix.

In some embodiments, a stereoisomer of formula I has the structure offormula I.b.1.i, I.b.1.ii, I.b.1.iii, I.b.1.iv, I.b.1.v, or I.b.1.vi. Insome embodiments, a stereoisomer of formula I has the structure offormula I.b.1.i. In some embodiments, a stereoisomer of formula I hasthe structure of formula I.b.1.ii. In some embodiments, a stereoisomerof formula I has the structure of formula I.b.1.iii. In someembodiments, a stereoisomer of formula I has the structure of formulaI.b.1.iv. In some embodiments, a stereoisomer of formula I has thestructure of formula I.b.1.v. In some embodiments, a stereoisomer offormula I has the structure of formula I.b.1.vi.

In some embodiments, a stereoisomer of formula I has the structure offormula I.b.2.i, I.b.2.ii, I.b.2.iii, I.b.2.iv, I.b.2.v, or I.b.2.vi. Insome embodiments, a stereoisomer of formula I has the structure offormula I.b.2.i. In some embodiments, a stereoisomer of formula I hasthe structure of formula I.b.2.ii. In some embodiments, a stereoisomerof formula I has the structure of formula I.b.2.iii. In someembodiments, a stereoisomer of formula I has the structure of formulaI.b.2.iv. In some embodiments, a stereoisomer of formula I has thestructure of formula I.b.2.v. In some embodiments, a stereoisomer offormula I has the structure of formula I.b.2.vi.

In some embodiments, the third threshold amount is 50%, 60%, 70%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% (w/w). In someembodiments, the third threshold amount is 50% (w/w). In someembodiments, the third threshold amount is 70% (w/w). In someembodiments, the third threshold amount is 80% (w/w). In someembodiments, the third threshold amount is 85% (w/w). In someembodiments, the third threshold amount is 90% (w/w). In someembodiments, the third threshold amount is 95% (w/w). In someembodiments, the third threshold amount is 96% (w/w). In someembodiments, the third threshold amount is 97% (w/w). In someembodiments, the third threshold amount is 98% (w/w). In someembodiments, the third threshold amount is 99% (w/w).

In some embodiments, the present invention provides a compositioncomprising one or more chemical entities of formula I, each of which isa compound of formula I:

-   a pharmaceutically acceptable salt thereof, a solvate thereof, or a    solvate of a pharmaceutically acceptable salt thereof,-   the composition characterized in that greater than a third threshold    amount of the total amount of chemical entities of formula I in the    composition are chemical entities of formula I.a.i:

-   wherein the third threshold amount is 50%.

In some embodiments, the present invention provides a compositioncomprising one or more chemical entities of formula I, each of which isa compound of formula I:

-   a pharmaceutically acceptable salt thereof, a solvate thereof, or a    solvate of a pharmaceutically acceptable salt thereof,-   the composition characterized in that greater than a third threshold    amount of the total amount of chemical entities of formula I in the    composition are chemical entities of formula I.a.ii:

-   wherein the third threshold amount is 50%.

In some embodiments, the present invention provides a compositioncomprising one or more chemical entities of formula I, each of which isa compound of formula I:

-   a pharmaceutically acceptable salt thereof, a solvate thereof, or a    solvate of a pharmaceutically acceptable salt thereof,-   the composition characterized in that greater than a third threshold    amount of the total amount of chemical entities of formula I in the    composition are chemical entities of formula I.b.1.i:

-   wherein the third threshold amount is 50%.

In some embodiments, the present invention provides a compositioncomprising one or more chemical entities of formula I, each of which isa compound of formula I:

-   a pharmaceutically acceptable salt thereof, a solvate thereof, or a    solvate of a pharmaceutically acceptable salt thereof,-   the composition characterized in that greater than a third threshold    amount of the total amount of chemical entities of formula I in the    composition are chemical entities of formula I.b.1.ii:

-   wherein the third threshold amount is 50%.

In some embodiments, the present invention provides a compositioncomprising one or more chemical entities of formula I, each of which isa compound of formula I:

-   a pharmaceutically acceptable salt thereof, a solvate thereof, or a    solvate of a pharmaceutically acceptable salt thereof,-   the composition characterized in that greater than a third threshold    amount of the total amount of chemical entities of formula I in the    composition are chemical entities of formula I.b.2.i:

-   wherein the third threshold amount is 50%.

In some embodiments, the present invention provides a compositioncomprising one or more chemical entities of formula I, each of which isa compound of formula I:

-   a pharmaceutically acceptable salt thereof, a solvate thereof, or a    solvate of a pharmaceutically acceptable salt thereof,-   the composition characterized in that greater than a third threshold    amount of the total amount of chemical entities of formula I in the    composition are chemical entities of formula I.b.2.ii:

-   wherein the third threshold amount is 50%.

In some embodiments, the second threshold amount is 50%, 60%, 70%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In someembodiments, the second threshold amount is 50%. In some embodiments,the second threshold amount is 70%. In some embodiments, the secondthreshold amount is 80%. In some embodiments, the second thresholdamount is 95%.

Liposomes for the Delivery of Agents, Such as mRNA

Among other things, the present invention provides compositioncomprising a lipid compound described herein for delivery of therapeuticagents. In some embodiments, a composition provided is a lipid basednanoparticle, such as a liposome. As used herein, the term “liposome”refers to any lamellar, multilamellar, or solid lipid nanoparticlevesicle. Typically, a liposome as used herein can be formed by mixingone or more lipids or by mixing one or more lipids and polymer(s). Thus,the term “liposome” as used herein encompasses both lipid and polymerbased nanoparticles. In particular, a liposome according to the presentinvention incorporates a lipid compound described herein as a cationiclipid component. As a non-limiting example, a liposome according to thepresent invention includes a composition comprising one or more chemicalentities of formula I. A suitable liposome may also contain second oradditional cationic lipids, helper lipids (e.g., non-cationic lipidsand/or cholesterol-based lipids), PEG-modified lipids, and/or polymers.

In some embodiments, cationic lipid(s) (e.g., the composition comprisingone or more chemical entities of formula I) constitute(s) about 30-50%(e.g., about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about35-40%) of the liposome by molar ratio. In some embodiments, thecationic lipid (e.g., the composition comprising one or more chemicalentities of formula I) constitutes about 30%, about 35%, about 40%,about 45%, or about 50% of the liposome by molar ratio. In someembodiments, the liposome comprises a second lipid or additionalcationic lipids.

Second or Additional Cationic Lipids

In some embodiments, liposomes may comprise a second or additionalcationic lipid. As used herein, the phrase “cationic lipid” refers toany of a number of lipid species that have a net positive charge at aselected pH, such as physiological pH. Several cationic lipids have beendescribed in the literature, many of which are commercially available.Particularly suitable cationic lipids for use in the compositions andmethods of the invention include those described in international patentpublications WO 2010/053572 (and particularly, C12-200 described atparagraph [00225]), WO 2012/170930 and WO 2013063468 each of which isincorporated herein by reference in its entirety. In certainembodiments, the compositions and methods of the invention employ alipid nanoparticles comprising an ionizable cationic lipid described inInternational Patent Application No. PCT/US2013/034602, filed Mar. 29,2013, Publ. No. WO 2013/149140 (incorporated herein by reference), suchas, e.g., (15Z, 18Z)—N,N-dimethyl-6-(9Z, 12Z)-octadeca-9,12-dien-1-yl)tetracosa-15,18-dien-1-amine (HGT5000), (15Z,18Z)—N,N-dimethyl-6-((9Z, 12Z)-octadeca-9,12-dien-1-yl)tetracosa-4,15,18-trien-1-amine (HGT5001), and(15Z,18Z)—N,N-dimethyl-6-((9Z, 12Z)-octadeca-9,12-dien-1-yl)tetracosa-5, 15, 18-trien-1-amine (HGT5002).

In some embodiments, the second or additional cationic lipidN-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride or “DOTMA”is used. (Feigner et al. (Proc. Nat'l Acad. Sci. 84, 7413 (1987); U.S.Pat. No. 4,897,355). DOTMA can be formulated alone or can be combinedwith the neutral lipid, dioleoylphosphatidyl-ethanolamine or “DOPE” orother cationic or non-cationic lipids into a liposomal transfer vehicleor a lipid nanoparticle, and such liposomes can be used to enhance thedelivery of nucleic acids into target cells. Other suitable cationiclipids include, for example, 5-carboxyspermylglycinedioctadecylamide or“DOGS,”2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminiumor “DOSPA” (Behr et al. Proc. Nat.'l Acad. Sci. 86, 6982 (1989); U.S.Pat. Nos. 5,171,678; 5,334,761), 1,2-Dioleoyl-3-Dimethylammonium-Propaneor “DODAP”, 1,2-Dioleoyl-3-Trimethylammonium-Propane or “DOTAP”.Additional exemplary cationic lipids also include1,2-distearyloxy-N,N-dimethyl-3-aminopropane or “DSDMA”,1,2-dioleyloxy-N,N-dimethyl-3-aminopropane or “DODMA”,1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane or “DLinDMA”,1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane or “DLenDMA”,N-dioleyl-N,N-dimethylammonium chloride or “DODAC”,N,N-distearyl-N,N-dimethylarnrnonium bromide or “DDAB”,N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide or “DMRIE”,3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propaneor “CLinDMA”, 2-[5′-(cholest-5-en-3-beta-oxy)-3′-oxapentoxy)-3-dimethy1-1-(cis,cis-9′, 1-2′-octadecadienoxy)propane or “CpLinDMA”,N,N-dimethyl-3,4-dioleyloxybenzylamine or “DMOBA”,1,2-N,N′-dioleylcarbamyl-3-dimethylaminopropane or “DOcarbDAP”,2,3-Dilinoleoyloxy-N,N-dimethylpropylamine or “DLinDAP”,1,2-N,N′-Dilinoleylcarbamyl-3-dimethylaminopropane or “DLincarbDAP”,1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane or “DLinCDAP”,2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane or “DLin-DMA”,2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane or“DLin-K-XTC2-DMA”, and2-(2,2-di((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethanamine(DLin-KC2-DMA)) (See, WO 2010/042877; Semple et al., Nature Biotech. 28:172-176 (2010)), or mixtures thereof. (Heyes, J., et al., J ControlledRelease 107: 276-287 (2005); Morrissey, D V., et al., Nat. Biotechnol.23(8): 1003-1007 (2005); PCT Publication WO2005/121348A1). In someembodiments, one or more of the cationic lipids comprise at least one ofan imidazole, dialkylamino, or guanidinium moiety.

In some embodiments, the second or additional cationic lipid may bechosen from XTC (2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane),MC3 (((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate), ALNY-100((3aR,5s,6aS)—N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine)), NC98-5(4,7,13-tris(3-oxo-3-(undecylamino)propyl)-N1,N16-diundecyl-4,7,10,13-tetraazahexadecane-1,16-diamide),DODAP (1,2-dioleyl-3-dimethylammonium propane), HGT4003 (WO 2012/170889,the teachings of which are incorporated herein by reference in theirentirety), ICE (WO 2011/068810, the teachings of which are incorporatedherein by reference in their entirety), HGT5000 (international patentpublication WO/2013/149140, the teachings of which are incorporatedherein by reference in their entirety) or HGT5001 (cis or trans)(WO/2013/149140), aminoalcohol lipidoids such as those disclosed inWO2010/053572, DOTAP (1,2-dioleyl-3-trimethylammonium propane), DOTMA(1,2-di-O-octadecenyl-3-trimethylammonium propane), DLinDMA (Heyes, J.;Palmer, L.; Bremner, K.; MacLachlan, I. “Cationic lipid saturationinfluences intracellular delivery of encapsulated nucleic acids” J.Contr. Rel. 2005, 107, 276-287), DLin-KC2-DMA (Semple, S. C. et al.“Rational Design of Cationic Lipids for siRNA Delivery” Nature Biotech.2010, 28, 172-176), C12-200 (Love, K. T. et al. “Lipid-like materialsfor low-dose in vivo gene silencing” PNAS 2010, 107, 1864-1869).

Non-Cationic/Helper Lipids

In some embodiments, provided liposomes contain one or more non-cationic(“helper”) lipids. As used herein, the phrase “non-cationic lipid”refers to any neutral, zwitterionic or anionic lipid. As used herein,the phrase “anionic lipid” refers to any of a number of lipid speciesthat carry a net negative charge at a selected H, such as physiologicalpH. Non-cationic lipids include, but are not limited to,distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine(DOPC), dipalmitoylphosphatidylcholine (DPPC),dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol(DPPG), dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), or a mixturethereof.

In some embodiments, such non-cationic lipids may be used alone, but arepreferably used in combination with other excipients, for example,cationic lipids. In some embodiments, the non-cationic lipid maycomprise a molar ratio of about 5% to about 90%, or about 10% to about70% of the total lipid present in a liposome. In some embodiments, anon-cationic lipid is a neutral lipid, i.e., a lipid that does not carrya net charge in the conditions under which the composition is formulatedand/or administered. In some embodiments, the percentage of non-cationiclipid in a liposome may be greater than 5%, greater than 10%, greaterthan 20%, greater than 30%, or greater than 40%.

Cholesterol-Based Lipids

In some embodiments, provided liposomes comprise one or morecholesterol-based lipids. For example, suitable cholesterol-basedcationic lipids include, for example, DC-Choi(N,N-dimethyl-N-ethylcarboxamidocholesterol),1,4-bis(3-N-oleylamino-propyl)piperazine (Gao, et al. Biochem. Biophys.Res. Comm. 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997);U.S. Pat. No. 5,744,335), or ICE. In some embodiments, thecholesterol-based lipid may comprise a molar ration of about 2% to about30%, or about 5% to about 20% of the total lipid present in a liposome.In some embodiments, The percentage of cholesterol-based lipid in thelipid nanoparticle may be greater than 5, %, 10%, greater than 20%,greater than 30%, or greater than 40%.

PEGylated Lipids

In some embodiments, provided liposomes comprise one or more PEGylatedlipids. For example, the use of polyethylene glycol (PEG)-modifiedphospholipids and derivatized lipids such as derivatized ceramides(PEG-CER), including N-Octanoyl-Sphingosine-1-[Succinyl(MethoxyPolyethylene Glycol)-2000] (C8 PEG-2000 ceramide) is also contemplatedby the present invention in combination with one or more of the cationicand, in some embodiments, other lipids together which comprise theliposome. Contemplated PEG-modified lipids include, but are not limitedto, a polyethylene glycol chain of up to 5 kDa in length covalentlyattached to a lipid with alkyl chain(s) of C₆-C₂₀ length. In someembodiments, a PEG-modified or PEGylated lipid is PEGylated cholesterolor PEG-2K. The addition of such components may prevent complexaggregation and may also provide a means for increasing circulationlifetime and increasing the delivery of the lipid-nucleic acidcomposition to the target cell, (Klibanov et al. (1990) FEBS Letters,268 (1): 235-237), or they may be selected to rapidly exchange out ofthe formulation in vivo (see U.S. Pat. No. 5,885,613).

In some embodiments, particularly useful exchangeable lipids arePEG-ceramides having shorter acyl chains (e.g., C₁₄ or C₁₈). ThePEG-modified phospholipid and derivitized lipids of the presentinvention may comprise a molar ratio from about 0% to about 15%, about0.5% to about 15%, about 1% to about 15%, about 4% to about 10%, orabout 2% of the total lipid present in the liposome.

According to various embodiments, the selection of second or additionalcationic lipids, non-cationic lipids and/or PEG-modified lipids whichcomprise the lipid nanoparticle, as well as the relative molar ratio ofsuch lipids to each other, is based upon the characteristics of theselected lipid(s), the nature of the intended target cells, thecharacteristics of the mRNA to be delivered. Additional considerationsinclude, for example, the saturation of the alkyl chain, as well as thesize, charge, pH, pKa, fusogenicity and toxicity of the selectedlipid(s). Thus the molar ratios may be adjusted accordingly. In someembodiments, the percentage of PEG-modified lipid in a liposome may begreater than 1%, greater than 2%, greater than 5%, greater than 10%, orgreater than 15%.

Polymers

In some embodiments, a suitable liposome according to the presentinvention further includes a polymer, in combination with one or morecationic lipids as described and, in some embodiments, other carriersincluding various lipids described herein. Thus, in some embodiments,liposomal delivery vehicles, as used herein, also encompass polymercontaining nanoparticles. Suitable polymers may include, for example,polyacrylates, polyalkycyanoacrylates, polylactide,polylactide-polyglycolide copolymers, polycaprolactones, dextran,albumin, gelatin, alginate, collagen, chitosan, cyclodextrins,protamine, PEGylated protamine, PLL, PEGylated PLL and polyethylenimine(PEI). When PEI is present, it may be branched PEI of a molecular weightranging from 10 to 40 kDA, e.g., 25 kDa branched PEI (Sigma #408727).

Therapeutic Agents

The present invention may be used to delivery any therapeutic agents.Specifically, any therapeutic agents to be administered to a subject maybe delivered using the complexes, picoparticles, nanoparticles,microparticles, micelles, or liposomes, described herein. The agent maybe an organic molecule (e.g., a therapeutic agent, a drug), inorganicmolecule, nucleic acid, protein, amino acid, peptide, polypeptide,polynucleotide, targeting agent, isotopically labeled organic orinorganic molecule, vaccine, immunological agent, etc. In certainembodiments of the present invention, the agent to be delivered may be amixture of agents.

In certain embodiments, the therapeutic agents are organic moleculeswith pharmaceutical activity, e.g., a drug. In certain embodiments, thedrug is an antibiotic, anti-viral agent, anesthetic, steroidal agent,anti-inflammatory agent, anti-neoplastic agent, anti-cancer agent,antigen, vaccine, antibody, decongestant, antihypertensive, sedative,birth control agent, progestational agent, anti-cholinergic, analgesic,anti-depressant, anti-psychotic, I3-adrenergic blocking agent, diuretic,cardiovascular active agent, vasoactive agent, non-steroidalanti-inflammatory agent, nutritional agent, etc.

Diagnostic agents include gases; metals; commercially available imagingagents used in positron emissions tomography (PET), computer assistedtomography (CAT), single photon emission computerized tomography, x-ray,fluoroscopy, and magnetic resonance imaging (MRI); and contrast agents.Examples of suitable materials for use as contrast agents in MRI includegadolinium chelates, as well as iron, magnesium, manganese, copper, andchromium. Examples of materials useful for CAT and x-ray imaging includeiodinebased materials.

Therapeutic and prophylactic agents include, but are not limited to,antibiotics, nutritional supplements, and vaccines. Vaccines maycomprise isolated proteins or peptides, inactivated organisms andviruses, dead organisms and viruses, genetically altered organisms orviruses, and cell extracts.

Polynucleotides

The present invention may be used to deliver any polynucleotide. Incertain embodiments, the polynucleotide is an interfering RNA (RNAi).The phenomenon of RNAi is discussed in greater detail, for example, inthe following references: Elbashir et al., 2001, Genes Dev., 15:188;Fire et al., 1998, Nature, 391:806; Tabara et al., 1999, Cell, 99:123;Hammond et al., Nature, 2000, 404:293; Zamore et al., 2000, Cell,101:25; Chakraborty, 2007, Curr. Drug Targets, 8:469; and Morris andRossi, 2006, Gene Ther., 13:553. In certain embodiments, thepolynucleotide is a dsRNA (double-stranded RNA). In certain embodiments,the polynucleotide is an siRNA (short interfering RNA). In certainembodiments, the polynucleotide is an shRNA (short hairpin RNA). Incertain embodiments, the polynucleotide is an miRNA (micro RNA). MicroRNAs (miRNAs) are genomically encoded non-coding RNAs of about 21-23nucleotides in length that help regulate gene expression, particularlyduring development. See, e.g., Bartel, 2004, Cell, 116:281; Novina andSharp, 2004, Nature, 430:161; and U.S. Patent Publication 2005/0059005;also reviewed in Wang and Li, 2007, Front. Biosci., 12:3975; and Zhao,2007, Trends Biochem. Sci., 32:189. In certain embodiments, thepolynucleotide is an antisense RNA.

In certain embodiments, the polynucleotide may be provided as anantisense agent or RNA interference (RNAi). See, e.g., Fire et al.,Nature 391:806-811, 1998. Antisense therapy is meant to include, e.g.,administration or in situ provision of single- or double-strandedoligonucleotides or their derivatives which specifically hybridize,e.g., bind, under cellular conditions, with cellular mRNA and/or genomicDNA, or mutants thereof, so as to inhibit expression of the encodedprotein, e.g., by inhibiting transcription and/or translation. See,e.g., Crooke “Molecular mechanisms of action of antisense drugs”Biochim. Biophys. Acta 1489(1):31-44, 1999; Crooke “Evaluating themechanism of action of antiproliferative antisense drugs” AntisenseNucleic Acid Drug Dev. 10(2):123-126, discussion 127, 2000; Methods inEnzymology volumes 313-314, 1999. The binding may be by conventionalbase pair complementarity, or, for example, in the case of binding toDNA duplexes, through specific interactions in the major groove of thedouble helix (i.e., triple helix formation). See, e.g., Chan et al., J.Mol. Med. 75(4):267-282, 1997.

In some embodiments, dsRNA, siRNA, shRNA, miRNA, antisense RNA, and/orRNAi can be designed and/or predicted using one or more of a largenumber of available algorithms. To give but a few examples, thefollowing resources can be utilized to design and/or predictpolynucleotides: algorithms found at Alnylum Online, Dharmacon Online,OligoEngine Online, Molecula Online, Ambion Online, BioPredsi Online,RNAi Web Online, Chang Bioscience Online, Invitrogen Online, LentiWebOnline GenScript Online, Protocol Online; Reynolds et al., 2004, Nat.Biotechnol., 22:326; Naito et al., 2006, Nucleic Acids Res., 34:W448; Liet al., 2007, RNA, 13:1765; Yiu et al., 2005, Bioinformatics, 21:144;and Jia et al., 2006, BMC Bioinformatics, 7: 271.

The polynucleotides may be of any size or sequence, and they may besingle- or double-stranded. In certain embodiments, the polynucleotideis greater than 100 base pairs long. In certain embodiments, thepolynucleotide is greater than 1000 base pairs long and may be greaterthan 10,000 base pairs long. The polynucleotide may be provided by anymeans known in the art. In certain embodiments, the polynucleotide hasbeen engineered using recombinant techniques. See, e.g., Ausubel et al.,Current Protocols in Molecular Biology (John Wiley & Sons, Inc., NewYork, 1999); Molecular Cloning: A Laboratory Manual, 2nd Ed., ed. bySambrook, Fritsch, and Maniatis (Cold Spring Harbor Laboratory Press:1989). The polynucleotide may also be obtained from natural sources andpurified from contaminating components found normally in nature. Thepolynucleotide may also be chemically synthesized in a laboratory. Incertain embodiments, the polynucleotide is synthesized using standardsolid phase chemistry.

The polynucleotide may be modified by chemical or biological means. Incertain embodiments, these modifications lead to increased stability ofthe polynucleotide. Modifications include methylation, phosphorylation,end-capping, etc.

mRNA

The present invention can be used to deliver any mRNA. mRNA is typicallythought of as the type of RNA that carries information from DNA to theribosome. The existence of mRNA is usually very brief and includesprocessing and translation, followed by degradation. Typically, ineukaryotic organisms, mRNA processing comprises the addition of a “cap”on the N-terminal (5′) end, and a “tail” on the C-terminal (3′) end. Atypical cap is a 7-methylguanosine cap, which is a guanosine that islinked through a 5′-5′-triphosphate bond to the first transcribednucleotide. The presence of the cap is important in providing resistanceto nucleases found in most eukaryotic cells. The tail is typically apolyadenylation event whereby a polyadenylyl moiety is added to the 3′end of the mRNA molecule. The presence of this “tail” serves to protectthe mRNA from exonuclease degradation. Messenger RNA typically istranslated by the ribosomes into a series of amino acids that make up aprotein.

Any mRNA capable of being translated into one or more peptides (e.g.,proteins) or peptide fragments is contemplated as within the scope ofthe present invention. In some embodiments, an mRNA encodes one or morenaturally occurring peptides. In some embodiments, an mRNA encodes oneor more modified or non-natural peptides.

In some embodiments an mRNA encodes an intracellular protein. In someembodiments, an mRNA encodes a cytosolic protein. In some embodiments,an mRNA encodes a protein associated with the actin cytoskeleton. Insome embodiments, an mRNA encodes a protein associated with the plasmamembrane. In some specific embodiments, an mRNA encodes a transmembraneprotein. In some specific embodiments an mRNA encodes an ion channelprotein. In some embodiments, an mRNA encodes a perinuclear protein. Insome embodiments, an mRNA encodes a nuclear protein. In some specificembodiments, an mRNA encodes a transcription factor. In someembodiments, an mRNA encodes a chaperone protein. In some embodiments,an mRNA encodes an intracellular enzyme (e.g., mRNA encoding an enzymeassociated with urea cycle or lysosomal storage metabolic disorders). Insome embodiments, an mRNA encodes a protein involved in cellularmetabolism, DNA repair, transcription and/or translation. In someembodiments, an mRNA encodes an extracellular protein. In someembodiments, an mRNA encodes a protein associated with the extracellularmatrix. In some embodiments an mRNA encodes a secreted protein. Inspecific embodiments, an mRNA used in the composition and methods of theinvention may be used to express functional proteins or enzymes that areexcreted or secreted by one or more target cells into the surroundingextracellular fluid (e.g., mRNA encoding hormones and/orneurotransmitters).

Synthesis of mRNA

mRNAs according to the present invention may be synthesized according toany of a variety of known methods. For example, mRNAs according to thepresent invention may be synthesized via in vitro transcription (IVT).Briefly, IVT is typically performed with a linear or circular DNAtemplate containing a promoter, a pool of ribonucleotide triphosphates,a buffer system that may include DTT and magnesium ions, and anappropriate RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase), DNAseI, pyrophosphatase, and/or RNAse inhibitor. The exact conditions willvary according to the specific application.

In some embodiments, for the preparation of mRNA according to theinvention, a DNA template is transcribed in vitro. A suitable DNAtemplate typically has a promoter, for example a T3, T7 or SP6 promoter,for in vitro transcription, followed by desired nucleotide sequence fordesired mRNA and a termination signal.

Desired mRNA sequence(s) according to the invention may be determinedand incorporated into a DNA template using standard methods. Forexample, starting from a desired amino acid sequence (e.g., an enzymesequence), a virtual reverse translation is carried out based on thedegenerated genetic code. Optimization algorithms may then be used forselection of suitable codons. Typically, the G/C content can beoptimized to achieve the highest possible G/C content on one hand,taking into the best possible account the frequency of the tRNAsaccording to codon usage on the other hand. The optimized RNA sequencecan be established and displayed, for example, with the aid of anappropriate display device and compared with the original (wild-type)sequence. A secondary structure can also be analyzed to calculatestabilizing and destabilizing properties or, respectively, regions ofthe RNA.

Modified mRNA

In some embodiments, mRNA according to the present invention may besynthesized as unmodified or modified mRNA. Typically, mRNAs aremodified to enhance stability. Modifications of mRNA can include, forexample, modifications of the nucleotides of the RNA. An modified mRNAaccording to the invention can thus include, for example, backbonemodifications, sugar modifications or base modifications. In someembodiments, mRNAs may be synthesized from naturally occurringnucleotides and/or nucleotide analogues (modified nucleotides)including, but not limited to, purines (adenine (A), guanine (G)) orpyrimidines (thymine (T), cytosine (C), uracil (U)), and as modifiednucleotides analogues or derivatives of purines and pyrimidines, such ase.g. 1-methyl-adenine, 2-methyl-adenine,2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine,N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine,4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine,1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine,7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil),dihydro-uracil, 2-thio-uracil, 4-thio-uracil,5-carboxymethylaminomethyl-2-thio-uracil,5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil,5-carboxy-methylaminomethyl-uracil, 5-methyl-2-thio-uracil,5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester,5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil,5′-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyaceticacid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil,queosine, 3-D-mannosyl-queosine, wybutosine, and phosphoramidates,phosphorothioates, peptide nucleotides, methylphosphonates,7-deazaguanosine, 5-methylcytosine and inosine. The preparation of suchanalogues is known to a person skilled in the art e.g. from the U.S.Pat. Nos. 4,373,071, 4,401,796, 4,415,732, 4,458,066, 4,500,707,4,668,777, 4,973,679, 5,047,524, 5,132,418, 5,153,319, 5,262,530 and5,700,642, the disclosures of which are incorporated by reference intheir entirety.

In some embodiments, mRNAs (e.g., enzyme encoding mRNAs) may contain RNAbackbone modifications. Typically, a backbone modification is amodification in which the phosphates of the backbone of the nucleotidescontained in the RNA are modified chemically. Exemplary backbonemodifications typically include, but are not limited to, modificationsfrom the group consisting of methylphosphonates, methylphosphoramidates,phosphoramidates, phosphorothioates (e.g. cytidine5′-O-(1-thiophosphate)), boranophosphates, positively chargedguanidinium groups etc., which means by replacing the phosphodiesterlinkage by other anionic, cationic or neutral groups.

In some embodiments, mRNAs (e.g., enzyme encoding mRNAs) may containsugar modifications. A typical sugar modification is a chemicalmodification of the sugar of the nucleotides it contains including, butnot limited to, sugar modifications chosen from the group consisting of2′-deoxy-2′-fluoro-oligoribonucleotide (2′-fluoro-2′-deoxycytidine5′-triphosphate, 2′-fluoro-2′-deoxyuridine 5′-triphosphate),2′-deoxy-2′-deamine-oligoribonucleotide (2′-amino-2′-deoxycytidine5′-triphosphate, 2′-amino-2′-deoxyuridine 5′-triphosphate),2′-O-alkyloligoribo-nucleotide, 2′-deoxy-2′-C-alkyloligoribonucleotide(2′-O-methylcytidine 5′-triphosphate, 2′-methyluridine 5′-triphosphate),2′-C-alkyloligoribonucleotide, and isomers thereof (2′-ara-cytidine5′-triphosphate, 2′-arauridine 5′-triphosphate), or azidotriphosphates(2′-azido-2′-deoxy-cytidine 5′-triphosphate, 2′-azido-2′-deoxyuridine5′-triphosphate).

In some embodiments, mRNAs (e.g., enzyme encoding mRNAs) may containmodifications of the bases of the nucleotides (base modifications). Amodified nucleotide which contains a base modification is also called abase-modified nucleotide. Exemples of such base-modified nucleotidesinclude, but are not limited to, 2-amino-6-chloropurine riboside5′-tri-phosphate, 2-aminoadenosine 5′-triphosphate, 2-thiocytidine5′-triphosphate, 2-thiouridine 5′-tri-phosphate, 4-thiouridine5′-triphosphate, 5-aminoallylcytidine 5′-triphosphate,5-aminoallyl-uridine 5′-triphosphate, 5-bromocytidine 5′-triphosphate,5-bromouridine 5′-triphosphate, 5-iodo-cytidine 5′-triphosphate,5-iodouridine 5′-triphosphate, 5-methylcytidine 5′-triphosphate,5-methyluridine 5′-triphosphate, 6-azacytidine 5′-triphosphate,6-azauridine 5′-triphosphate, 6-chloropurine riboside 5′-triphosphate,7-deazaadenosine 5′-triphosphate, 7-deazaguanosine 5′-triphosphate,8-azaadenosine 5′-triphosphate, 8-azidoadenosine 5′-triphosphate,benzimidazole riboside 5′-triphosphate, N1-methyladenosine5′-triphosphate, N1-methylguanosine 5′-tri-phosphate, N6-methyladenosine5′-triphosphate, 06-methylguanosine 5′-triphosphate, pseudo-uridine5′-triphosphate, puromycin 5′-triphosphate or xanthosine5′-triphosphate.

Cap Structure

Typically, mRNA synthesis includes the addition of a “cap” on theN-terminal (5′) end, and a “tail” on the C-terminal (3′) end. Thepresence of the cap is important in providing resistance to nucleasesfound in most eukaryotic cells. The presence of a “tail” serves toprotect the mRNA from exonuclease degradation.

Thus, in some embodiments, mRNAs (e.g., enzyme encoding mRNAs) include a5′ cap structure. A 5′ cap is typically added as follows: first, an RNAterminal phosphatase removes one of the terminal phosphate groups fromthe 5′ nucleotide, leaving two terminal phosphates; guanosinetriphosphate (GTP) is then added to the terminal phosphates via aguanylyl transferase, producing a 5′5′5 triphosphate linkage; and the7-nitrogen of guanine is then methylated by a methyltransferase.Examples of cap structures include, but are not limited to, m7G(5′)ppp(5′(A,G(5′)ppp(5′)A and G(5′)ppp(5′)G.

In some embodiments, naturally occurring cap structures comprise a7-methyl guanosine that is linked via a triphosphate bridge to the5′-end of the first transcribed nucleotide, resulting in a dinucleotidecap of m⁷G(5′)ppp(5′)N, where N is any nucleoside. In vivo, the cap isadded enzymatically. The cap is added in the nucleus and is catalyzed bythe enzyme guanylyl transferase. The addition of the cap to the 5′terminal end of RNA occurs immediately after initiation oftranscription. The terminal nucleoside is typically a guanosine, and isin the reverse orientation to all the other nucleotides, i.e.,G(5′)ppp(5′)GpNpNp.

A common cap for mRNA produced by in vitro transcription ism⁷G(5′)ppp(5′)G, which has been used as the dinucleotide cap intranscription with T7 or SP6 RNA polymerase in vitro to obtain RNAshaving a cap structure in their 5′-termini. The prevailing method forthe in vitro synthesis of capped mRNA employs a pre-formed dinucleotideof the form m⁷G(5′)ppp(5′)G (“m⁷GpppG”) as an initiator oftranscription.

To date, a usual form of a synthetic dinucleotide cap used in in vitrotranslation experiments is the Anti-Reverse Cap Analog (“ARCA”) ormodified ARCA, which is generally a modified cap analog in which the 2′or 3′ OH group is replaced with —OCH₃.

Additional cap analogs include, but are not limited to, chemicalstructures selected from the group consisting of m⁷GpppG, m⁷GpppA,m⁷GpppC; unmethylated cap analogs (e.g., GpppG); dimethylated cap analog(e.g., m^(2,7)GpppG), trimethylated cap analog (e.g., m2,2,7GpppG),dimethylated symmetrical cap analogs (e.g., m⁷Gpppm⁷G), or anti reversecap analogs (e.g., ARCA; m^(7,2′Ome)GpppG, m^(72′d)GpppG,m^(7,3′Ome)GpppG, m^(7,3′d)GpppG and their tetraphosphate derivatives)(see, e.g., Jemielity, J. et al., “Novel ‘anti-reverse’ cap analogs withsuperior translational properties”, RNA, 9: 1108-1122 (2003)).

In some embodiments, a suitable cap is a 7-methyl guanylate (“m⁷G”)linked via a triphosphate bridge to the 5′-end of the first transcribednucleotide, resulting in m⁷G(5′)ppp(5′)N, where N is any nucleoside. Apreferred embodiment of a m⁷G cap utilized in embodiments of theinvention is m⁷G(5′)ppp(5′)G.

In some embodiments, the cap is a Cap0 structure. Cap0 structures lack a2′-O-methyl residue of the ribose attached to bases 1 and 2. In someembodiments, the cap is a Cap1 structure. Cap1 structures have a2′-O-methyl residue at base 2. In some embodiments, the cap is a Cap2structure. Cap2 structures have a 2′-O-methyl residue attached to bothbases 2 and 3.

A variety of m⁷G cap analogs are known in the art, many of which arecommercially available. These include the m⁷GpppG described above, aswell as the ARCA 3′-OCH₃ and 2′-OCH₃ cap analogs (Jemielity, J. et al.,RNA, 9: 1108-1122 (2003)). Additional cap analogs for use in embodimentsof the invention include N7-benzylated dinucleoside tetraphosphateanalogs (described in Grudzien, E. et al., RNA, 10: 1479-1487 (2004)),phosphorothioate cap analogs (described in Grudzien-Nogalska, E., etal., RNA, 13: 1745-1755 (2007)), and cap analogs (including biotinylatedcap analogs) described in U.S. Pat. Nos. 8,093,367 and 8,304,529,incorporated by reference herein.

Tail Structure

Typically, the presence of a “tail” serves to protect the mRNA fromexonuclease degradation. The poly A tail is thought to stabilize naturalmessengers and synthetic sense RNA. Therefore, in certain embodiments along poly A tail can be added to an mRNA molecule thus rendering the RNAmore stable. Poly A tails can be added using a variety of art-recognizedtechniques. For example, long poly A tails can be added to synthetic orin vitro transcribed RNA using poly A polymerase (Yokoe, et al. NatureBiotechnology. 1996; 14: 1252-1256). A transcription vector can alsoencode long poly A tails. In addition, poly A tails can be added bytranscription directly from PCR products. Poly A may also be ligated tothe 3′ end of a sense RNA with RNA ligase (see, e.g., Molecular CloningA Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis(Cold Spring Harbor Laboratory Press: 1991 edition)).

In some embodiments, mRNAs (e.g., enzyme encoding mRNAs) include a 3′poly(A) tail structure. Typically, the length of the poly A tail can beat least about 10, 50, 100, 200, 300, 400 at least 500 nucleotides (SEQID NO: 1). In some embodiments, a poly-A tail on the 3′ terminus of mRNAtypically includes about 10 to 300 adenosine nucleotides (e.g., about 10to 200 adenosine nucleotides, about 10 to 150 adenosine nucleotides,about 10 to 100 adenosine nucleotides, about 20 to 70 adenosinenucleotides, or about 20 to 60 adenosine nucleotides). In someembodiments, mRNAs include a 3′ poly(C) tail structure. A suitablepoly-C tail on the 3′ terminus of mRNA typically include about 10 to 200cytosine nucleotides (SEQ ID NO: 2) (e.g., about 10 to 150 cytosinenucleotides, about 10 to 100 cytosine nucleotides, about 20 to 70cytosine nucleotides, about 20 to 60 cytosine nucleotides, or about 10to 40 cytosine nucleotides). The poly-C tail may be added to the poly-Atail or may substitute the poly-A tail.

In some embodiments, the length of the poly A or poly C tail is adjustedto control the stability of a modified sense mRNA molecule of theinvention and, thus, the transcription of protein. For example, sincethe length of the poly A tail can influence the half-life of a sensemRNA molecule, the length of the poly A tail can be adjusted to modifythe level of resistance of the mRNA to nucleases and thereby control thetime course of polynucleotide expression and/or polypeptide productionin a target cell.

5′ and 3′ Untranslated Region

In some embodiments, mRNAs include a 5′ and/or 3′ untranslated region.In some embodiments, a 5′ untranslated region includes one or moreelements that affect an mRNA's stability or translation, for example, aniron responsive element. In some embodiments, a 5′ untranslated regionmay be between about 50 and 500 nucleotides in length.

In some embodiments, a 3′ untranslated region includes one or more of apolyadenylation signal, a binding site for proteins that affect anmRNA's stability of location in a cell, or one or more binding sites formiRNAs. In some embodiments, a 3′ untranslated region may be between 50and 500 nucleotides in length or longer.

Exemplary 3′ and/or 5′ UTR sequences can be derived from mRNA moleculeswhich are stable (e.g., globin, actin, GAPDH, tubulin, histone, orcitric acid cycle enzymes) to increase the stability of the sense mRNAmolecule. For example, a 5′ UTR sequence may include a partial sequenceof a CMV immediate-early 1 (IE1) gene, or a fragment thereof to improvethe nuclease resistance and/or improve the half-life of thepolynucleotide. Also contemplated is the inclusion of a sequenceencoding human growth hormone (hGH), or a fragment thereof to the 3′ endor untranslated region of the polynucleotide (e.g., mRNA) to furtherstabilize the polynucleotide. Generally, these modifications improve thestability and/or pharmacokinetic properties (e.g., half-life) of thepolynucleotide relative to their unmodified counterparts, and include,for example modifications made to improve such polynucleotides'resistance to in vivo nuclease digestion.

According to various embodiments, any size mRNA may be encapsulated byprovided liposomes. In some embodiments, the provided liposomes mayencapsulate mRNA of greater than about 0.5 kb, 1 kb, 1.5 kb, 2 kb, 2.5kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, or 5 kb in length.

Liposomes

The liposomes for use in provided compositions can be prepared byvarious techniques which are presently known in the art. For example,multilamellar vesicles (MLV) may be prepared according to conventionaltechniques, such as by depositing a selected lipid on the inside wall ofa suitable container or vessel by dissolving the lipid in an appropriatesolvent, and then evaporating the solvent to leave a thin film on theinside of the vessel or by spray drying. An aqueous phase may then addedto the vessel with a vortexing motion which results in the formation ofMLVs. Uni-lamellar vesicles (ULV) can then be formed by homogenization,sonication or extrusion of the multi-lamellar vesicles. In addition,unilamellar vesicles can be formed by detergent removal techniques.

In certain embodiments, provided compositions comprise a liposomewherein an agent, such as a nucleic acid e.g., mRNA, is associated onboth the surface of the liposome and encapsulated within the sameliposome. For example, during preparation of the compositions of thepresent invention, cationic liposomes may associate with the mRNAthrough electrostatic interactions. For example, during preparation ofthe compositions of the present invention, cationic liposomes mayassociate with the mRNA through electrostatic interactions.

In some embodiments, the compositions and methods of the inventioncomprise mRNA encapsulated in a liposome. In some embodiments, the oneor more mRNA species may be encapsulated in the same liposome. In someembodiments, the one or more mRNA species may be encapsulated indifferent liposomes. In some embodiments, the mRNA is encapsulated inone or more liposomes, which differ in their lipid composition, molarratio of lipid components, size, charge (Zeta potential), targetingligands and/or combinations thereof. In some embodiments, the one ormore liposome may have a different composition of cationic lipids,neutral lipid, PEG-modified lipid and/or combinations thereof. In someembodiments the one or more lipisomes may have a different molar ratioof cationic lipid, neutral lipid, cholesterol and PEG-modified lipidused to create the liposome.

The process of incorporation of a desired therapeutic agent, such as anucleic acid (e.g., mRNA), into a liposome is often referred to as“loading”. Exemplary methods are described in Lasic, et al., FEBS Lett.,312: 255-258, 1992, which is incorporated herein by reference. Theliposome-incorporated nucleic acids may be completely or partiallylocated in the interior space of the liposome, within the bilayermembrane of the liposome, or associated with the exterior surface of theliposome membrane. The incorporation of a nucleic acid into liposomes isalso referred to herein as “encapsulation” wherein the nucleic acid isentirely contained within the interior space of the liposome. Thepurpose of incorporating a mRNA into a transfer vehicle, such as aliposome, is often to protect the nucleic acid from an environment whichmay contain enzymes or chemicals that degrade nucleic acids and/orsystems or receptors that cause the rapid excretion of the nucleicacids. Accordingly, in some embodiments, a suitable delivery vehicle iscapable of enhancing the stability of the mRNA contained therein and/orfacilitate the delivery of mRNA to the target cell or tissue.

Liposome Size

Suitable liposomes in accordance with the present invention may be madein various sizes. In some embodiments, provided liposomes may be madesmaller than previously known mRNA encapsulating liposomes. In someembodiments, decreased size of liposomes is associated with moreefficient delivery of mRNA. Selection of an appropriate liposome sizemay take into consideration the site of the target cell or tissue and tosome extent the application for which the liposome is being made.

In some embodiments, an appropriate size of liposome is selected tofacilitate systemic distribution of antibody encoded by the mRNA. Insome embodiments, it may be desirable to limit transfection of the mRNAto certain cells or tissues. For example, to target hepatocytes aliposome may be sized such that its dimensions are smaller than thefenestrations of the endothelial layer lining hepatic sinusoids in theliver; in such cases the liposome could readily penetrate suchendothelial fenestrations to reach the target hepatocytes.

Alternatively or additionally, a liposome may be sized such that thedimensions of the liposome are of a sufficient diameter to limit orexpressly avoid distribution into certain cells or tissues. For example,a liposome may be sized such that its dimensions are larger than thefenestrations of the endothelial layer lining hepatic sinusoids tothereby limit distribution of the liposomes to hepatocytes.

In some embodiments, a suitable liposome has a size of or less thanabout 500 nm, 450 nm, 400 nm, 350 nm, 300 nm, 250 nm, 200 nm, 150 nm,125 nm, 110 nm, 100 nm, 95 nm, 90 nm, 85 nm, 80 nm, 75 nm, 70 nm, 65 nm,60 nm, 55 nm, or 50 nm. In some embodiments, a suitable liposome has asize no greater than about 250 nm (e.g., no greater than about 225 nm,200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, or 50 nm). In someembodiments, a suitable liposome has a size ranging from about 10-250 nm(e.g., ranging from about 10-225 nm, 10-200 nm, 10-175 nm, 10-150 nm,10-125 nm, 10-100 nm, 10-75 nm, or 10-50 nm). In some embodiments, asuitable liposome has a size ranging from about 100-250 nm (e.g.,ranging from about 100-225 nm, 100-200 nm, 100-175 nm, 100-150 nm). Insome embodiments, a suitable liposome has a size ranging from about10-100 nm (e.g., ranging from about 10-90 nm, 10-80 nm, 10-70 nm, 10-60nm, or 10-50 nm).

A variety of alternative methods known in the art are available forsizing of a population of liposomes. One such sizing method is describedin U.S. Pat. No. 4,737,323, incorporated herein by reference. Sonicatinga liposome suspension either by bath or probe sonication produces aprogressive size reduction down to small ULV less than about 0.05microns in diameter. Homogenization is another method that relies onshearing energy to fragment large liposomes into smaller ones. In atypical homogenization procedure, MLV are recirculated through astandard emulsion homogenizer until selected liposome sizes, typicallybetween about 0.1 and 0.5 microns, are observed. The size of theliposomes may be determined by quasi-electric light scattering (QELS) asdescribed in Bloomfield, Ann. Rev. Biophys. Bioeng., 10:421-150 (1981),incorporated herein by reference. Average liposome diameter may bereduced by sonication of formed liposomes. Intermittent sonicationcycles may be alternated with QELS assessment to guide efficientliposome synthesis.

Pharmaceutical Compositions

To facilitate delivery of an agent, such as a nucleic acid e.g., mRNA,and/or expression of mRNA in vivo, delivery vehicles such as liposomescan be formulated in combination with one or more additional nucleicacids, carriers, targeting ligands or stabilizing reagents, or inpharmacological compositions where it is mixed with suitable excipients.Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition.

Provided liposomally-encapsulated agents, such as a nucleic acid e.g.,mRNA and compositions containing the same, may be administered and dosedin accordance with current medical practice, taking into account theclinical condition of the subject, the site and method ofadministration, the scheduling of administration, the subject's age,sex, body weight and other factors relevant to clinicians of ordinaryskill in the art. The “effective amount” for the purposes herein may bedetermined by such relevant considerations as are known to those ofordinary skill in experimental clinical research, pharmacological,clinical and medical arts. In some embodiments, the amount administeredis effective to achieve at least some stabilization, improvement orelimination of symptoms and other indicators as are selected asappropriate measures of disease progress, regression or improvement bythose of skill in the art. For example, a suitable amount and dosingregimen is one that causes at least transient protein (e.g., enzyme)production.

Suitable routes of administration include, for example, oral, rectal,vaginal, transmucosal, pulmonary including intratracheal or inhaled, orintestinal administration; parenteral delivery, including intradermal,transdermal (topical), intramuscular, subcutaneous, intramedullaryinjections, as well as intrathecal, direct intraventricular,intravenous, intraperitoneal, and/or intranasal administration.

Alternately or additionally, liposomally encapsulated agents, such as anucleic acid e.g., mRNA and compositions of the invention may beadministered in a local rather than systemic manner, for example, viainjection of the pharmaceutical composition directly into a targetedtissue, preferably in a sustained release formulation. Local deliverycan be affected in various ways, depending on the tissue to be targeted.For example, aerosols containing compositions of the present inventioncan be inhaled (for nasal, tracheal, or bronchial delivery);compositions of the present invention can be injected into the site ofinjury, disease manifestation, or pain, for example; compositions can beprovided in lozenges for oral, tracheal, or esophageal application; canbe supplied in liquid, tablet or capsule form for administration to thestomach or intestines, can be supplied in suppository form for rectal orvaginal application; or can even be delivered to the eye by use ofcreams, drops, or even injection. Formulations containing providedcompositions complexed with therapeutic molecules or ligands can even besurgically administered, for example in association with a polymer orother structure or substance that can allow the compositions to diffusefrom the site of implantation to surrounding cells. Alternatively, theycan be applied surgically without the use of polymers or supports.

In some embodiments, provided liposomes and/or compositions areformulated such that they are suitable for extended-release of theagent, e.g., mRNA contained therein. Such extended-release compositionsmay be conveniently administered to a subject at extended dosingintervals. For example, in one embodiment, the compositions of thepresent invention are administered to a subject twice day, daily orevery other day. In a preferred embodiment, the compositions of thepresent invention are administered to a subject twice a week, once aweek, every ten days, every two weeks, every three weeks, or morepreferably every four weeks, once a month, every six weeks, every eightweeks, every other month, every three months, every four months, everysix months, every eight months, every nine months or annually. Alsocontemplated are compositions and liposomes which are formulated fordepot administration (e.g., intramuscularly, subcutaneously,intravitreally) to either deliver or release a mRNA over extendedperiods of time. Preferably, the extended-release means employed arecombined with modifications made to the mRNA to enhance stability.

Also contemplated herein are lyophilized pharmaceutical compositionscomprising one or more of the liposomes disclosed herein and relatedmethods for the use of such compositions as disclosed for example, inInternational Patent Application No. PCT/US2012/041663, filed Jun. 8,2012, Publ. No. WO 2012/170889, the teachings of which are incorporatedherein by reference in their entirety. For example, lyophilizedpharmaceutical compositions according to the invention may bereconstituted prior to administration or can be reconstituted in vivo.For example, a lyophilized pharmaceutical composition can be formulatedin an appropriate dosage form (e.g., an intradermal dosage form such asa disk, rod or membrane) and administered such that the dosage form isrehydrated over time in vivo by the individual's bodily fluids.

Provided liposomes and compositions may be administered to any desiredtissue. In some embodiments, the agent, e.g., mRNA delivered by providedliposomes or compositions is expressed in the tissue in which theliposomes and/or compositions were administered. In some embodiments,the mRNA delivered is expressed in a tissue different from the tissue inwhich the liposomes and/or compositions were administered Exemplarytissues in which delivered mRNA may be delivered and/or expressedinclude, but are not limited to the liver, kidney, heart, spleen, serum,brain, skeletal muscle, lymph nodes, skin, and/or cerebrospinal fluid.

According to various embodiments, the timing of expression of deliveredagents, e.g., mRNAs, can be tuned to suit a particular medical need. Insome embodiments, the expression of the protein encoded by deliveredmRNA is detectable 1, 2, 3, 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66,and/or 72 hours in serum or target tissues after a single administrationof provided liposomes or compositions. In some embodiments, theexpression of the protein encoded by the mRNA is detectable 1 day, 2days, 3 days, 4 days, 5 days, 6 days, and/or 7 days in serum or targettissues after a single administration of provided liposomes orcompositions. In some embodiments, the expression of the protein encodedby the mRNA is detectable 1 week, 2 weeks, 3 weeks, and/or 4 weeks inserum or target tissues after a single administration of providedliposomes or compositions. In some embodiments, the expression of theprotein encoded by the mRNA is detectable after a month or longer aftera single administration of provided liposomes or compositions.

EXAMPLES Example 1—Synthesis of Racemic Compounds of Formula I

Racemic compound 10 was prepared from protected lysine derivative 1,Boc-Lys(Z)-OSu, by cleavage of the alpha-amino tert-butyl carbamate withtrifluoroacetic acid and dimerization of the resulting free amine toform 3,6-dioxopiperazine 2. Hydrogenation of 2 over catalytic palladiumyields primary diamine 3, which is treated with four equivalents ofepoxide 4 and triethylamine to form the racemic 3,6-dioxopiperazine 10of formula I.

Dibenzyl(((2S,5S)-3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl))dicarbamate2: To Boc-Lys(Z)-OSu 1 (50 g) cooled with an ice bath was added TFA (60mL) slowly. The resulting mixture was stirred for 20 min. The ice bathwas removed and stirring was continued for 1 h. TFA was removed byrotary evaporation. The oily residue was dissolved in DMF (80 mL) andadded slowly to stirred pyridine (anhydrous, 2.5 L). Mass spectrometryindicated completion of reaction after 1 h. Pyridine was removed byrotary evaporation and the residue was diluted with EtOAc (2 L). After20 min of stirring the mixture was filtered to give 2 as off-white solid(20 g after overnight vacuum drying. Yield: 73%).

(3S,6S)-3,6-bis(4-Aminobutyl)piperazine-2,5-dione-2HOAc 3: To compound 2in a mixture of AcOH (550 mL) and DCM (550 mL) was added Pd/C (10%, wet.10 g). This mixture was stirred under a H₂ balloon for 4 h when massspectrometry indicated completion of reaction. The reaction mixture wasflushed with nitrogen for 10 min and filtered through Celite. The Celitewas rinsed with MeOH (3×250 mL). The combined filtrate was evaporatedand the residue was stirred with EtOAc (1.0 L) for 30 min. Filtrationgave 3 as white solid (16.37 g after overnight vacuum drying. Yield:114%. ¹H NMR shows clean product but with extra HOAc in sample)

3,6-bis(4-(bis(2-Hydroxydodecyl)amino)butyl)piperazine-2,5-dione 10: Toa mixture of 3 (15.87 g, 42.2 mmol) and 4 (57 mL, 261 mmol) in EtOH (75mL) stirred in a 500 mL pressure flask at room temperature was addedEt₃N (23 mL, 165 mmol). The flask was flushed with nitrogen for 5 minand sealed. The mixture (solid and liquid slurry) was stirred for 30 minat room temperature then heated to 150-155° C. (oil bath temperature)and stirred for 5 h. A clear solution was obtained after temperaturereached 150° C. After being cooled to room temperature the reactionsolution was evaporated and the residue was purified by flash columnchromatography 9 times with 0-30% MeOH/DCM as eluent and 2 times with0-30% MeOH/EtOAc as eluent. Use of DCM to 50% of 75:22:3 DCM/MeOH/NH4OH(aq.) as eluent led to co-elution of product with a less polar sideproduct. The side product ran closely with product on TLC with 30%MeOH/DCM as developing solvents. It was well separated from product onTLC with 30% MeOH/EtOAc as developing solvents. Pure product fractionsfrom column purifications were pooled and evaporated. The oily residuewas dissolved in Et₂O and evaporated. Drying under vacuum overnightremoved all solvents and gave the racemic compound 10 as light yellowgel (9.85 g, yield: 24%). HPLC with ELSD detection showed two similarsized peaks with same product mass. Elemental Analysis: (Calc): C,72.63; H, 12.17; N, 5.64; (Obsd): C, 72.25; H, 12.37; N, 5.68. MassSpec: 993.8 m/z.

In another run with 7.15 g of 3 and 25.6 mL of 4 the crude product waspurified twice with 0-30% MeOH/EtOAc as eluent to give two batches ofproduct: the 1.55 g batch from early fractions and the 7.38 g batch fromlate fractions. Both batches were pure by ¹H NMR. HPLC with ELSDdetection showed two similar sized peaks with same product mass for the7.38 g batch but only a single product peak for the 1.55 g batch.

Example 2—Synthesis of Chiral Compounds of Formula I.b.1 (i.e., Compound10)

Chiral compound 10 was synthesized via N-alkylation of protected lysinederivative 5 with two equivalents of epoxide 4 to form diol 6.Hydrogenation of diol 6 over catalytic palladium forms alpha amino acid7, which is divided into two portions. The first portion of alpha aminoacid 7 has its alpha amino group protected as the tert-butyl carbamate,upon treatment with Boc anhydride, to form carboxylic acid 8. The secondportion of alpha amino acid 7 has its free acid converted to the methylester to form amine 9. Carboxylic acid 8 and amine 9 are coupled to anamide intermediate via peptide coupling reagents such as HATU anddiethanolamine in aprotic solvent such as DMF, the tert-butyl carbamategroup of the amide intermediate is cleaved with trifluoroacetic acid indichloromethane, and the resulting amino ester product is cyclized tothe piperazine-2,5-dione 10. The stereochemistry at all chiral centersis preserved via this route.

Chiral compounds 12-15, below, are prepared via described syntheticroutes using respective chiral epoxide starting materials.

Example 3—the Compound of Formula I.a.i (i.e., R4-SR-cKK-E12)

Synthesis of compound 3.1: To a suspension of Mg (60 g, 2.5 mol) inanhydrous Et₂O (1000 mL) was added one crystal of iodine, followed byaddition of 1-bromononane (25 mL). The reaction was initiated and thesolution began to reflux after heating the reaction flask with a waterbath. The remaining 1-bromononane (360 mL, 2.0 mol total) was addedthrough an additional funnel in 1.5 h to maintain the reflux. Afteraddition, the reaction solution was heated at reflux with a water bathfor an additional 30 min and then it was cooled to room temperature.This ether solution of 3.1 was used directly in the next reaction.

Synthesis of compound 4.1: To a suspension of CuI (39 g, 0.2 mol) inanhydrous THF (1000 mL) stirred with a mechanical stirrer at −78° C. ina 5 L three-necked flask was added R-epichlorohydrin (185 g, 2.0 mol)(additional funnel). After addition, the above ether solution of 3.1 wasadded via a cannula in 1 h. The mixture was stirred at −78° C. for 3.5hrs, then quenched with saturated aqueous NH₄Cl (1500 mL). The organiclayer was separated. The aqueous layer was extracted with Et₂O (2000mL).The combined organic phase was washed with brine, dried (overMgSO₄), and evaporated under vacuum. The crude product was purified byflash column chromatography (2.5 kg SiO₂, eluted with 0-10% EtOAc inHexanes) to give 292 g of 4.1 (yield: 66%) as a light yellow oil.

Synthesis of compound 5.1: To a solution of 4.1 (292 g, 1.33 mol) inMeOH (600 mL) and THF (600 mL) was added aqueous NaOH (1.5M, 1000 mL)through an additional funnel at 0° C. After addition the reactionmixture was stirred at room temperature for 2.5 hrs, TLC showed a majorproduct, very minor amount of starting material (EtOAc:Hexanes=1:9,Rf=0.6). THF and MeOH were removed by rotary evaporation under vacuum.The aqueous residue was extracted with Et₂O (600 mL×3). The combinedorganic phase was washed with brine, dried over MgSO₄, and evaporated.The yellow oily residue was purified by column (SiO₂, 2.5 kg, elutedwith 0-10% EtOAc in hexanes) to afford 205 g (84%) of pure 5.1.

Synthesis of compound 7.1: Method A: To a solution of 6.1 (75 g, 0.25mol) in a mixture of DCM (1000 mL) and MeOH (71 mL) that was beingstirred at room temperature was added aqueous Na₂CO₃ (2.0 M, 135 mL).Organic layer was separated and the aqueous layer was extracted with DCM(250 mL×2). The combined organic phase was dried over Na₂SO₄ andevaporated under vacuum. The residue was dissolved in MeOH (375 mL),then compound 5.1 (185 g, 1.0 mol) was added. The reaction mixture wasstirred at room temperature for 4 days when MS and TLC showed mostlydesired product. After concentration, the crude product was purified byflash column chromatography (2.0 kg SiO₂, eluted with 0-60% EtOAc inhexanes) to give 7.1 (131 g, 82%) as a pale viscous oil.

Method B: To a suspension of 8.1 (50 g, 0.2 mol) in MeOH (600 mL) wasadded DIPEA (45 mL), then compound 5.1 (150 g, 0.813 mol, 4.0 equiv) wasadded. The reaction mixture was stirred at room temperature for 7 days.Solvents were removed, the residue was purified by column (1.0 kg ofSiO₂, eluted with 0-30% MeOH in EtOAc) to give 9.1 as a waxy solid (83g, 67%). To a solution of 9.1 (81 g, 0.13 mol) in DMF (1000 mL) stirredat 0° C. was added HATU (50.1 g, 0.13 mol), followed by DIPEA (92 mL,0.52 mol). The mixture was stirred at 0° C. for 40 min, and then MeOH(53.2 mL, 10.0 equiv) was added. The resulting mixture was stirred atroom temperature overnight. It was then diluted with water (5000 mL) andextracted with EtOAc (500 mL×4). The combined organic phase was washedwith brine (600 mL×3), dried over anhydrous MgSO₄ and evaporated undervacuum. The crude product was purified by column (1.0 kg of SiO₂, elutedwith 0-80% EtOAc in hexanes) to give 7.1 (69 g, 55% for 2 steps) as apale viscous oil.

Synthesis of compound 10.1: To a solution of 7.1 (69 g, 0.11 mol) in DCM(200 mL) was added TFA (200 mL), the mixture was stirred at roomtemperature for 2 hrs, MS detection showed only desired product. Allsolvents were evaporated under vacuum to give 115 g of a brown coloredoil-like product 10.1, which was used in the next step without furtherpurification.

Synthesis of compound 12.1: To a suspension of 11.1, Boc-D-lysine (75 g,0.305 mol) in MeOH (900 mL) were added DIPEA (68 mL) and 5.1 (196 g,1.06 mol). The mixture was stirred at room temperature for 7 days.Volatiles were removed and the crude product was purified by column (2.5kg of SiO₂, eluted with 0-40% MeOH in EtOAc) to give 118 g (63%) of purecompound 12.1.

Synthesis of compound 13.1: To a solution of 12.1 (67.5 g, 0.11 mol) inDMF (600 mL, warm up to 50° C. for 30 min to obtain a homogeneoussolution) that was cooled with an ice-bath were added HATU (50 g, 0.12mol) and DIPEA (95 mL, 0.55 mol). The resulting mixture was stirred at0° C. for 45 min, then compound 10.1 (115 g, obtained above) in DMF (400mL) was added using an additional funnel. The mixture was stirred atroom temperature overnight. Ether (1000 mL) was added, followed by water(1000 mL). The organic phase was separated; the aqueous was extractedwith ether (250 mL×2). The combined organic phase was washed with water,dried over anhydrous MgSO₄, filtered and concentrated. The residue waspurified by column (1.0 kg of SiO₂, eluted with 0-20% MeOH in EtOAc) togive 94.2 g (76%) of compound 13.1.

Synthesis of the compound of Formula I.a.i (i.e., R4-SR-cKK-E12): To asolution of 13.1 (94 g, 0.084 mol) in DCM (300 mL) was added TFA (300mL). The mixture was stirred at room temperature for 2 hrs. MS detectionshowed complete reaction. Solvents were evaporated under vacuum. Theresidue was dissolved in DCM (500 mL) and washed with aqueous Na₂CO₃(1.0 M, 500 mL). The aqueous wash was back extracted with DCM (100 mL).The combined organic phase was dried over anhydrous NaSO₄ andevaporated. The residue was dissolved in MeOH (1500 mL) and cooled withan ice-bath. Aqueous NH₄OH (28%, 80 mL) was added through additionalfunnel. The reaction mixture was allowed to slowly warm up to roomtemperature and stirred at ambient temperature for 2 days. Volatileswere evaporated under vacuum. The crude product was purified by column(1.0 kg of SiO₂, eluted with solvents: 1% NH₄OH, 4-9% MeOH, 95-90%EtOAc) to give 34 g of pure R4-SR-cKK-E12 and 22 g of impureR4-SR-cKK-E12. The impure R4-SR-cKK-E12 was re-purified by column togive 12 g of pure R4-SR-cKK-E12. Thus, a total 46 g (55%) of pureR4-SR-cKK-E12 (gummy solid) was obtained.

Example 4—the Compound of Formula I.a.ii (i.e., S4-SR-cKK-E12)

Synthesis of compound 3.1: To a suspension of Mg (30 g, 1.25 mol) inanhydrous Et₂O (600 mL) was added one crystal of iodine, followed byaddition of 1-bromononane (30 mL). The reaction was initiated and thesolution began to reflux after heating the reaction flask with a waterbath. The remaining 1-bromononane (161 mL, 2.0 mol total) was addedthrough an additional funnel in 1.5 h to maintain the reflux. Afteraddition, the reaction solution was heated at reflux with a water bathfor an additional 30 min and then it was cooled to room temperature.This ether solution of 3.1 was used directly in the next reaction.

Synthesis of compound 4.2: To a suspension of CuI (19 g, 0.1 mol) inanhydrous THF (1000 mL) that was being stirred with a mechanical stirrerat −78° C. in a 5 L three-necked flask was added S-epichlorohydrin (92g, 1.0 mol) using an additional funnel. After addition, the above ethersolution of 3.2 was added via a cannula in 1 h. The mixture was stirredat −78° C. for 3.5 hrs, then quenched with saturated aqueous NH₄Cl (400mL). The organic layer was separated. The aqueous layer was extractedwith Et₂O (1000 mL).The combined organic phase was washed with brine,dried (over MgSO₄), and evaporated under vacuum. The crude product waspurified by flash column chromatography (2.5 kg SiO₂, eluted with 0-10%EtOAc in hexanes) to give 111.6 g of 4.2 (yield: 66%) as a light yellowoil.

Synthesis of compound 5.2: To a solution of 4.2 (111.3 g, 0.506 mol) inMeOH (230 mL) and THF (230 mL) was added aqueous NaOH (1.5M, 395 mL)using an additional funnel at 0° C. After addition the reaction mixturewas stirred at room temperature for 2.5 hrs, TLC showed a major productand very minor amount of starting material (EtOAc:Hexanes=1:9, Rf=0.6).THF and MeOH were removed by rotary evaporation under vacuum. Theaqueous residue was extracted with Et₂O (200 mL×3). The combined organicphase was washed with brine, dried over MgSO₄, and evaporated. Theyellow oily residue was purified by column (SiO₂, 1.0 kg, eluted with0-10% EtOAc in hexanes) to afford 81 g (87%) of pure 5.2.

Synthesis of compound 7.2: To a solution of 6.1 (13 g, 0.044 mol) in amixture of DCM (100 mL) and MeOH (10 mL) that was being stirred at roomtemperature was added aqueous Na₂CO₃ (2.0 M, 25 mL). Organic layer wasseparated and the aqueous layer was extracted with DCM (250 mL×2). Thecombined organic phase was dried over Na₂SO₄ and evaporated undervacuum. The residue was dissolved in MeOH (60 mL), then compound 5.2 (32g, 0.174 mol) was added. The reaction mixture was stirred at roomtemperature for 4 days when MS and TLC showed mostly desired product.After concentration, the crude product was purified by flash columnchromatography (600 g SiO₂, eluted with 0-60% EtOAc in hexanes) to give7.2 (23 g, 85%) as a pale viscous oil.

Synthesis of compound 8.2: To a solution of 7.2 (23 g, 0.0366 mol) inDCM (60 mL) was added TFA (60 mL), the mixture was stirred at roomtemperature for 2 hrs, MS detection showed only desired product. Allsolvents were evaporated under vacuum to give 40 g of a brown coloredoil-like product 8.2, which was used in the next step without furtherpurification.

Synthesis of compound 10.2: To a suspension of 11.1, Boc-D-lysine (14 g,0.057 mol) in MeOH (900 mL) were added TEA (11.6 mL) and 5.2 (42 g,0.228 mol). The mixture was stirred at room temperature for 7 days.Volatiles were removed and the crude product was purified by column (1.0kg of SiO₂, eluted with 0-40% MeOH in EtOAc) to give 24 g (70%) of purecompound 10.2.

Synthesis of compound 11.2: To a solution of 10.2 (9.1 g, 14.82 mmol) inDMF (120 mL) that was being cooled with an ice bath were added HATU (8.4g, 22.23 mol) and DIPEA (25 mL, 148.2 mmol). The mixture was stirred at0° C. for 45 min, then compound 8.2 in DMF (80 mL) was added using anadditional funnel. The resulting mixture was stirred at room temperatureovernight. MS detection showed no starting material. Ether (1000 mL) wasadded, followed by water (1000 mL). The organic phase was separated, theaqueous was extracted with ether (200 mL×2). The combined organic phasewas washed with brine, dried with anhydrous MgSO₄, filtered andconcentrated. The residue was purified by column (330 g of SiO₂, elutedwith 0-20% MeOH in EtOAc) to give 10.6 g of desired compound 11.2.

Synthesis of the compound of Formula I.a.ii (i.e., S4-SR-cKK-E12): To asolution of 11.2 (10.6 g, 0.084 mol) in DCM (30 mL) was added TFA (30mL). The mixture was stirred at room temperature for 2 hrs. MS detectionshowed complete reaction. Solvents were evaporated under vacuum. Theresidue was dissolved in DCM (150 mL) and washed with aqueous Na₂CO₃(1.0 M, 200 mL). The aqueous wash was back extracted with DCM (100 mL).The combined organic phase was dried over anhydrous NaSO₄ andevaporated. The residue was dissolved in MeOH (200 mL) and cooled withan ice-bath. Aqueous NH₄OH (28%, 10 mL) was added using an additionalfunnel. The reaction mixture was allowed to slowly warm up to roomtemperature and stirred at ambient temperature for 2 days. Volatileswere evaporated under vacuum. The crude product was purified by column(600 g of SiO₂, eluted with solvents: 1% NH₄OH, 4-9% MeOH, 95-90% EtOAc)give 5.1 g of pure S4-SR-cKK-E12.

Example 5—the Compound of Formula I.b.1.i (i.e., R4-SS-cKK-E12)

Synthesis of compound 3.3(N²-(tert-butoxycarbonyl)-N⁶,N⁶-bis((R)-2-hydroxydodecyl)-L-lysine): Amixture of R-epoxide (5.1, 46 g, 250 mmol), Boc-L-Lysine 8.1 (15 g, 61mmol), and diisopropylethylamine (11 ml) in methanol (80 ml) was stirredat room temperature for 3 d. Volatiles were removed and the yellow oilyresidue was purified by chromatography on silica gel (330 g) elutingwith EtOAc/MeOH (100/0 to 70/30, 20 min) to give product 3.3 as a whitesolid (9.7 g, 26%).

Synthesis of the compound of Formula I.b.1.i. (i.e., R4-SS-cKK-E12;((3S,6S)-3,6-bis(4-(bis((S)-2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione)):To a solution of 3.3 (7.4 g, 12 mmol) and NHS (1.38 g, 12 mmol) in DCM(280 ml) was added DCC (2.97 g, 14.4 mmol). The reaction mixture wasstirred at room temperature for 1.5 h. The solvent was then removed andthe residue (crude 4.3) was dissolved in TFA (30 ml). The resultingmixture was stirred at room temperature for 1 h. TFA was then removedand DCM (30 ml) was added to the residue, and co-evaporated to removeresidual TFA. The crude 5.3 was dissolved in DCM (30 mL) and added toanhydrous pyridine (480 mL) at 0° C. under N2. The resulting mixture wasstirred at room temperature overnight. Pyridine was then removed and theresidue was diluted with diethyl ether (300 mL). The white solid formedwas removed by filtration. The filtrate was washed with aqueous Na₂CO₃(1M, 150 ml) and brine (150 ml), dried over Na₂SO₄, and concentrated.The residue was purified by column chromatography five times (one 330 gcolumn followed by four 80 g column) eluting with 3% NH₄OH/7% MeOH/90%EtOAc to give 1.05 g of pure R4-SS-cKK-E12 as pale gum. 1.0 g ofR4-SS-cKK-E12.

Example 6—the Compound of Formula I.b.1.ii (i.e., S4-SS-cKK-E12)

Synthesis of compound 3(N²-(tert-butoxycarbonyl)-N⁶,N⁶-bis((S)-2-hydroxydodecyl)-L-lysine): Amixture of S-epoxide (5.2, 46 g, 250 mmol), Boc-L-Lysine 8.1 (15 g, 61mmol) and diisopropylethylamine (11 ml) in methanol (80 ml) was stirredat room temperature for 8 d. Volatiles were removed and the yellow oilyresidue was purified by chromatography on silica gel (330 g) elutingwith EtOAc/MeOH (100/0 to 70/30, 20 min) to give product 3.4 as a whitesolid (22 g, 59%).

Synthesis of the compound of Formula I.b.1.ii (i.e., S4-SS-cKK-E12;((3S,6S)-3,6-bis(4-(bis((S)-2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione):To a solution of 3.4 (24.5 g, 40 mmol) and NHS (4.6 g, 40 mmol) in DCM(280 ml) was added DCC (9.9 g, 48 mmol). The reaction mixture wasstirred at room temperature for 3 h. The solvent was then removed andthe residue (crude 4.4) was dissolved in TFA (100 ml) at 0° C. Theresulting mixture was allowed to warm up to room temperature and stirredfor 45 min. TFA was then removed and DCM (120 ml) was added to theresidue, and then co-evaporated to remove residual TFA. The crude 5.4was dissolved in anhydrous pyridine (1.6 L) at 0° C. under N2, and theresulting mixture was stirred at room temperature overnight. Thepyridine was then removed by rotavap, and the residue was diluted withdiethyl ether (1 L). The white solid formed was removed by filtrationand washed with diethyl ether (200 ml). The filtrate was washed withaqueous Na₂CO₃ (1M, 500 ml) and brine (500 ml), dried over Na₂SO₄, andconcentrated. The residue was purified by column chromatography onsilica gel (330 g) eluting with 0-50% (3% NH₄OH/7% MeOH/90% EtOAc)/EtOActo give 6.8 g TLC pure S4-SS-cKK-E12 and 7.1 g slightly impureS4-SS-cKK-E12. The 6.8 g (6.8 mmol) TLC pure S4-SS-cKK-E12 was dissolvedin 120 ml of ethyl acetate. Boc₂O (0.22 g, 1.0 mmol) was added. Themixture was stirred at room temperature for 2 h. The solvent was removedand the residue was purified by column chromatography on silica gel (120g) eluting with 0-50% (3% NH₄OH/7% MeOH/90% EtOAc)/EtOAc to give 5.7 g(84%) of pure product S4-SS-cKK-E12 which was free of an amine sideproduct.

Example 7—the Compound of Formula I.b.2.i (i.e., R4-RR-cKK-E12)

Synthesis of compound 3.1: To magnesium (60 g) suspended in anhydrousEt₂O (0.9 L) was added 1-bromononane 2.1 (20 mL), followed by additionof a catalytic amount of iodine (50 mg). The resulting mixture washeated with a hot water bath until reaction of Mg with 2.1 started. Thebath was removed and the remaining 1-bromononane (379.1 mL) was added tomaintain a gentle reflux. After addition of 2.1 the reflux wasmaintained by a hot water bath for another 30 min. The resultingGrignard solution of 3.1 was cooled and used directly in next step.

Synthesis of compound 4.1: To CuI (38.1 g) suspended in THF (1.5 L) at−78° C. was added R-(−)-epichlorohydrin (156.8 mL). Then the aboveGrignard solution of 3.1 was added via a cannula while the reactiontemperature was maintained at <−65° C. The resulting reaction mixturewas stirred at −78° C. for an additional 3 hour. Then, saturated aqueousammonium chloride solution (0.8 L) was added carefully, followed byaddition of water (1.0 L). The resulting mixture was stirred and allowedto warm up to room temperature. The organic layer was separated, and theaqueous layer was extracted with Et₂O (0.5 L×2). The organic layer wascombined with the Et₂O extracts and the resulting solution was washedwith brine (0.5 L×2), and dried over anhydrous magnesium sulfate.Solvents were evaporated under vacuum, and the resulting residue waspurified on a silica gel column (hexanes to 20% EtOAc/hexanes) toprovide 4.1 (243.5 g, 55%) as a yellow oil.

Synthesis of compound 5.1: To a solution of 4.1 (243.49 g) in 1:2.6MeOH-THF (3.6 L) stirred at 0° C. was slowly added aqueous NaOH solution(1.5 M, 0.89 L, 1.33 mole). The resulting mixture was allowed to warm upand stirred at room temperature for 3 h. TLC analysis showed completedisappearance of 4.1. Organic solvents were evaporated, and the aqueouslayer was extracted with Et₂O (1 L+500 mL×2). The organic extracts werecombined, washed with brine (600 mL), and dried over anhydrous magnesiumsulfate. Solvents were evaporated under vacuum to give a residue whichwas purified on silica gel column (hexanes to 10% EtOAc/hexanes) toprovide the epoxide 5.1 (193.7 g, 95%) as a light yellow oil.

Synthesis of compound 12.1: A mixture of N-Boc-D-Lysine 11.1 (49.2 g,0.2 mole) and the epoxide 5.1 (147.2 g, 0.8 mole) in MeOH (1.04 L) wasstirred at room temperature. DIPEA (51.9 g, 0.4 mole) was added. Theresulting mixture was then stirred for 8 days, and then concentrated todryness. The residue was purified on a silica gel column (2 kg,MeOH/DCM, 0 to 10%) to give 49.2 g of mostly pure 12.1 (MZ-550-180) and58.3 g of impure 12.1, which was purified by a second column (1.5 kg,MeOH/EtOAc, 10 to 40%) to give 41.4 g of mostly pure 12.1. The twomostly pure batches were combined and stirred with EtOAc (0.5 L) for 3h. The mixture was filtered to give 41.4 g pure 12.1 as a white solid.The filtrate was concentrated to dryness. The residue was stirred withEtOAc (0.1 L) for 1 h and filtered to give 10.6 g of pure 12.1. Thefiltrate was concentrated to dryness and the residue was purified on asilica gel column (330 g, MeOH/EtOAc, 10 to 40%) to give a third batchof 26.9 g pure 12.1. A total of 78.9 g of pure 12.1 was obtained. Yield:64%

Preparation of the compound of Formula I.b.2.i (i.e., R4-RR-cKK-E12):Batch 1: To a solution of 12.1 (6.14 g, 10 mmol) and N-hydroxysuccimide(1.15 g, 10 mmol) in DCM (70 mL) was added DCC (2.47 g, 12 mmol). Theresulting mixture was stirred at room temperature for 3 h. Volatileswere evaporated under vacuum to give a residue (NHS ester 6.5), whichwas dissolved in TFA (25 mL) and stirred for 0.5 h. TFA was removedunder vacuum, and the residue (compound 7.5) was cooled to 0° C.Pyridine (anhydrous, 400 mL) was added, and the reaction mixture wasstirred at room temperature for 2 h. Pyridine was removed under vacuum,and the residue was suspended in Et₂O (300 mL). The solid was removed byfiltration. The filtrate was washed with 1 M Na₂CO₃ aqueous solution(150 mL) and brine (150 mL), dried over anhydrous magnesium sulfate, andconcentrated to dryness. The residue was separated by columnchromatograph (80 g, 7:3 MeOH—NH₃—H₂O (4× with EtOAc)/EtOAc, 0 to 50%)to give R4-RR-cKK-E12 as gummy solid (2.22 g). Multiple precipitationsand triturations from EtOAc gave pure R4-RR-cKK-E12 (0.46 g) as a gum.

Example 8—the Compound of Formula I.b.2.ii (i.e., S4-RR-cKK-E12)

Synthesis of compound 3.1: To magnesium (60 g) suspended in anhydrousEt₂O (0.9 L) was added 1-bromononane 2.1 (20 mL), followed by additionof a catalytic amount of iodine (50 mg). The resulting mixture washeated with a hot water bath until reaction of Mg with 2.1 started. Thebath was removed and the remaining 1-bromononane (379.1 mL) was added tomaintain a gentle reflux. After addition of 2.1 the reflux wasmaintained by a hot water bath for another 30 min. The resultingGrignard solution of 3.1 was cooled and used directly in next step.

Synthesis of compound 4.2: To CuI (38.1 g) suspended in THF (1.5 L) at−78° C. was added S-(−)-epichlorohydrin (156.8 mL). Then the aboveGrignard solution of 3.1 was added via a cannula while the reactiontemperature was maintained at <−65° C. The resulting reaction mixturewas stirred at −78° C. for an additional 3 hour. Then, saturated aqueousammonium chloride solution (0.8 L) was added carefully, followed byaddition of water (1.0 L). The resulting mixture was stirred and allowedto warm up to room temperature. The organic layer was separated, and theaqueous layer was extracted with Et₂O (0.5 L×2). The organic layer wascombined with the Et₂O extracts and the resulting solution was washedwith brine (0.5 L×2), and dried over anhydrous magnesium sulfate.Solvents were evaporated under vacuum, and the residue was purified on asilica gel column (hexanes to 20% EtOAc/hexanes) to provide 4.2 (250.8g, 57%) as a light yellow oil.

Synthesis of compound 5.2: To a solution of 4.2 (250.8 g) in 1:2.6MeOH-THF (3.9 L) stirred at 0° C. was added slowly aqueous NaOH solution(1.5 M, 1.36 mole, 0.90 L). The resulting mixture was allowed to warm upand stirred at room temperature for 3 h. TLC analysis showed completedisappearance of 4.2. Organic solvents were evaporated, and the aqueouslayer was extracted with Et₂O (1 L+500 mL×2). The organic extracts werecombined, washed with brine (600 mL), and dried over anhydrous magnesiumsulfate. Solvents were evaporated under vacuum to give a residue whichwas purified on silica gel column (hexanes to 10% EtOAc/hexanes) toprovide 5.2 (195.4 g, 93%) as a light yellow oil.

A mixture of N-Boc-D-Lysine 11.1 (49.2 g, 0.2 mole) and the epoxide 5.2(147.2 g, 0.8 mole) in MeOH (1.04 L) was stirred at room temperature.DIPEA (51.9 g, 0.4 mole) was added. The resulting mixture was thenstirred for 8 days, and then concentrated to dryness. The resultingresidue was purified on a silica gel column (2 kg, MeOH/EtOAc, 10 to30%) to give 10.2 (79.9 g, 65%) as a white solid.

Preparation of the compound of Formula I.b.2.ii (i.e., S4-RR-cKK-E12) Toa solution of 10.2 (61.4 g, 100 mmol) and N-hydroxysuccimide (11.5 g,100 mmol) in DCM (800 mL) was added DCC (24.7 g, 120 mmol). Theresulting mixture was stirred at room temperature for 4 h. Volatileswere evaporated under vacuum to give a residue (NHS ester 6.6), whichwas dissolved in TFA (25 mL) and stirred for 40 min. TFA was removedunder vacuum, and the residue (compound 7.6) was cooled to 0° C.Pyridine (anhydrous, 3.5 L) was added, and the reaction mixture wasstirred at room temperature for 19 h. Pyridine was removed under vacuum,and the residue was suspended in Et₂O (3.0 L). The solid was removed byfiltration. The filtrate was washed with 1 M aqueous Na₂CO₃ solution(1.0 L) and brine (1.0 L), dried over anhydrous magnesium sulfate, andconcentrated to dryness. The residue was purified by columnchromatograph (4×330 g silica gel column eluting with (7% MeOH/3%NH₃—H₂O/90% EtOAc)/EtOAc, 0 to 50%) to provide S4-RR-cKK-E12 as a gummysolid (15.9 g, 16%)

Example 9—Formulations

The formulations described herein consisted of a multi-component lipidmixture of varying ratios employing one or more cationic lipids, helperlipids and PEGylated lipids designed to encapsulate various nucleicacid-based materials. The cationic lipid utilized throughout is thecompound of formula I(3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione).Helper lipids can include (but not exclusively) DSPC(1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC(1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE(1,2-dioleyl-sn-glycero-3-phosphoethanolamine), DOPC(1,2-dioleyl-sn-glycero-3-phosphotidylcholine) DPPE(1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE(1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG(,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol)), cholesterol, etc.The PEGylated lipids can include (but not exclusively) a poly(ethylene)glycol chain of up to 5 kDa in length covalently attached to a lipidwith alkyl chain(s) of C₆-C₂₀ length.

Messenger RNA Material

Human Factor IX (FIX), codon-optimized Firefly Luciferase (FFL) andcodon-optimized human argininosuccinate synthetase (ASS1) messenger RNAwere synthesized by in vitro transcription from a plasmid DNA templateencoding the gene, which was followed by the addition of a 5′ capstructure (Cap 1) (Fechter, P.; Brownlee, G. G. “Recognition of mRNA capstructures by viral and cellular proteins” J. Gen. Virology 2005, 86,1239-1249) and a 3′ poly(A) tail of approximately 250 nucleotides (SEQID NO: 3) in length as determined by gel electrophoresis. 5′ and 3′untranslated regions present in each mRNA product are represented as Xand Y, respectively and defined as stated (vide infra).

Codon-Optirnized Human Argininosuccinate Synthetase (ASS1) rnRNA:(SEO ID NO: 4) XAUGAGCAGCAAGGGCAGCGUGGUGCUGGCCUACAGCGGCGGCCUGGACACCAGCUGCAUCCUGGUGUGGCUGAAGGAGCAGGGCUACGACGUGAUCGCCUACCUGGCCAACAUCGGCCAGAAGGAGGACUUCGAGGAGGCCCGCAAGAAGGCCCUGAAGCUGGGCGCCAAGAAGGUGUUCAUCGAGGACGUGAGCCGCGAGUUCGUGGAGGAGUUCAUCUGGCCCGCCAUCCAGAGCAGCGCCCUGUACGAGGACCGCUACCUGCUGGGCACCAGCCUGGCCCGCCCCUGCAUCGCCCGCAAGCAGGUGGAGAUCGCCCAGCGCGAGGGCGCCAAGUACGUGAGCCACGGCGCCACCGGCAAGGGCAACGACCAGGUGCGCUUCGAGCUGAGCUGCUACAGCCUGGCCCCCCAGAUCAAGGUGAUCGCCCCCUGGCGCAUGCCCGAGUUCUACAACCGCUUCAAGGGCCGCAACGACCUGAUGGAGUACGCCAAGCAGCACGGCAUCCCCAUCCCCGUGACCCCCAAGAACCCCUGGAGCAUGGACGAGAACCUGAUGCACAUCAGCUACGAGGCCGGCAUCCUGGAGAACCCCAAGAACCAGGCCCCCCCCGGCCUGUACACCAAGACCCAGGACCCCGCCAAGGCCCCCAACACCCCCGACAUCCUGGAGAUCGAGUUCAAGAAGGGCGUGCCCGUGAAGGUGACCAACGUGAAGGACGGCACCACCCACCAGACCAGCCUGGAGCUGUUCAUGUACCUGAACGAGGUGGCCGGCAAGCACGGCGUGGGCCGCAUCGACAUCGUGGAGAACCGCUUCAUCGGCAUGAAGAGCCGCGGCAUCUACGAGACCCCCGCCGGCACCAUCCUGUACCACGCCCACCUGGACAUCGAGGCCUUCACCAUGGACCGCGAGGUGCGCAAGAUCAAGCAGGGCCUGGGCCUGAAGUUCGCCGAGCUGGUGUACACCGGCUUCUGGCACAGCCCCGAGUGCGAGUUCGUGCGCCACUGCAUCGCCAAGAGCCAGGAGCGCGUGGAGGGCAAGGUGCAGGUGAGCGUGCUGAAGGGCCAGGUGUACAUCCUGGGCCGCGAGAGCCCCCUGAGCCUGUACAACGAGGAGCUGGUGAGCAUGAACGUGCAGGGCGACUACGAGCCCACCGACGCCACCGGCUUCAUCAACAUCAACAGCCUGCGCCUGAAGGAGUACCACCGCCUGCAGAGCAAGGUGACCGCCAAGUGAY 5′ and 3′ UTR Sequences (SEO ID NO: 5)X = GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACG (SEO ID NO: 6)Y = CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUC

Aliquots of 50 mg/mL ethanolic solutions of one or more compounds offormula I, DOPE, Cholesterol and DMG-PEG2K were mixed and diluted withethanol to 3 mL final volume. Separately, an aqueous buffered solution(10 mM citrate/150 mM NaCl, pH 4.5) of ASS1 mRNA was prepared from a 1mg/mL stock. The lipid solution was injected rapidly into the aqueousmRNA solution and shaken to yield a final suspension in 20% ethanol. Theresulting nanoparticle suspension was filtered, diafiltrated with 1×PBS(pH 7.4), concentrated and stored at 2-8° C. Final concentration=0.64mg/mL ASS1 mRNA (encapsulated). Z_(ave)=78 nm (Dv₍₅₀₎=46 nm; Dv₍₉₀₎=96nm).

Example 10—Analysis of ASS1 Protein Produced Via Intravenously DeliveredASS1 mRNA-Loaded Nanoparticles

Injection Protocol

All studies were performed using male CD-1 mice of approximately 6-8weeks of age at the beginning of each experiment. Samples wereintroduced by a single bolus tail-vein injection of an equivalent totaldose of 1.0 mg/kg (or otherwise specified) of encapsulated ASS1 mRNA.Mice were sacrificed and perfused with saline at the designated timepoints.

Isolation of Organ Tissues for Analysis

The liver, spleen, kidney and heart of each mouse was harvested,apportioned into separate parts, and stored in either 10% neutralbuffered formalin or snap-frozen and stored at −80° C. for analysis.

Isolation of Plasma for Analysis

All animals were euthanized by CO₂ asphyxiation at designated timepoints post dose administration (±5%) followed by thoracotomy andterminal cardiac blood collection. Whole blood (maximal obtainablevolume) will be collected via cardiac puncture on euthanized animalsinto serum separator tubes, allowed to clot at room temperature for atleast 30 minutes, centrifuged at 22° C.±5° C. at 9300 g for 10 minutes,and the serum will be extracted. For interim blood collections,approximately 40-50 μL of whole blood will be collected via facial veinpuncture or tail snip. Samples collected from non treatment animals wereused as a baseline ASS1 levels for comparison to study animals.

Enzyme-Linked Immunosorbent Assay (ELISA) Analysis

Human ASS1 ELISA: Standard ELISA procedures were followed employingmouse anti-ASS1 2D1-2E12 IgG as the capture antibody with rabbitanti-ASS1 #3285 IgG as the secondary (detection) antibody (Shire HumanGenetic Therapies). Horseradish peroxidase (HRP)-conjugated goatanti-rabbit IgG was used for activation of the3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution. The reactionwas quenched using 2N H₂SO₄ after 20 minutes. Detection was monitoredvia absorption (450 nm) on a Molecular Device SpectraMax instrument.Untreated mouse serum and organs and human ASS1 protein were used asnegative and positive controls, respectively.

Example 11—In Vivo Human ASS1 Protein Production

The production of human ASS1 protein via codon-optimized hASS1mRNA-loaded lipid nanoparticles, comprising compounds of formula I, wastested in CD-1 mice as a single, bolus intravenous injection, asdescribed above. FIG. 1 depicts the amount of human ASS1 proteindetected via ELISA when treating mice human ASS1 mRNA-loaded lipidnanoparticles, with various racemic and chiral compounds of formula I,at 1.0 mg/kg doses. The mice were sacrificed twenty-four hourspost-injection and organs, such as livers, were harvested.

Example 12—Toxicity Studies

Expression levels of alanine aminotransferase (ALT) and aspartateaminotransferase (AST) were measured for various racemic and chiralcompounds of formula I. Increased expression levels of AST and/or ALTgenerally caused by agents that cause liver toxicity. The chiralcompounds of formula I generally yielded lower expression levels of ALTand/or AST, i.e., correlating to lower liver toxicity issues, comparedto stereochemically non-enriched, or stereochemically less enriched,compositions of the same lipid. See Tables 1 and 2 below.

TABLE 1 ASS1 (ng/mg Total Structure ALT (U/L) AST (U/L) Protein) RacemicMixture 190 ± 43  212 ± 54  471 ± 309 201 ± 89  403 ± 42  937 ± 337 207± 84  425 ± 169 497 ± 213 344 ± 57  555 ± 122 1387 ± 593  426 ± 112 757± 158 1509 ± 598  457 ± 274 728 ± 126 910 ± 327 503 ± 201 653 ± 133 1010± 154  618 ± 503 638 ± 273 209 ± 169 S₄ with 170 ± 40  132 ± 44  375 ±244 Racemic Lysine Core 155 ± 57  157 ± 38  674 ± 147 R₄ with 188 ± 22 265 ± 122 823 ± 215 Racemic Lysine Core 236 ± 163 237 ± 139 568 ± 248Racemic —OH with 378 ± 58  622 ± 76  117 ± 80  SS Lysine Core 618 ± 503638 ± 273 209 ± 169 S₄-S,S-cKKE12 226 ± 71  384 ± 233 1121 ± 468 R₄-S,S-cKKE12 175 ± 102 144 ± 35  449 ± 105 S₄-S,R-cKKE12 190 ± 75  193± 71  2303 ± 491  R₄-S,R-cKKE12 75 ± 13 82 ± 12 264 ± 317 86 ± 27 119 ±32  1369 ± 233  94 ± 22 88 ± 16 467 ± 149 59 ± 13 73 ± 18 401 ± 137 139± 28  177 ± 73  1182 ± 150  180 ± 19  141 ± 25  750 ± 324 269 ± 80  424± 156 2790 ± 464  123 ± 39  124 ± 22  1113 ± 35  60 ± 4  49 ± 5  846 ±226 70 ± 10 78 ± 24 1082 ± 189 

TABLE 2 cKK-E12 of a single intravenous dose. 24 hours post- formulationused for screening was cKK-E12:DOPE:Chol:DMG- Structure PEG2K AssignmentALT AST Lot #1 ‘Racemic’ 885 ± 489 982 ± 350 Mixture 207 ± 84  425 ± 169504 ± 317 657 ± 176 503 ± 201 653 ± 133 Lot #2 365 ± 152 604 ± 136 401 ±265 586 ± 193 Lot #3 197 ± 50  309 ± 33  Lot #1 S₄-SS 226 ± 71  384 ±233 Lot #1 R₄-SS 175 ± 102 144 ± 35  Lot #1 S₄-RR 152 ± 9  180 ± 42  Lot#1 R₄-RR 136 ± 34  194 ± 80  Lot #1 S₄-RS/SR 143 ± 29  240 ± 98  189 ±47  203 ± 87  Lot #2 190 ± 75  193 ± 71  Lot #1 R₄-RS/SR 86 ± 27 119 ±32  75 ± 13 82 ± 12 76 ± 4  79 ± 4  94 ± 22 88 ± 16 Lot #2 269 ± 80  424± 156 139 ± 28  177 ± 73  180 ± 19  141 ± 25  91 ± 13 98 ± 18 Lot #3 125± 47  104 ± 27  Lot #4 94 ± 24 91 ± 14 Lot #5 60 ± 4  49 ± 5  Lot #6 70± 10 78 ± 24 Lot #7 308 ± 115 354 ± 128 123 ± 39  124 ± 22 

While several embodiments of the present invention have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, kit, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,kits, and/or methods, if such features, systems, articles, materials,kits, and/or methods are not mutually inconsistent, is included withinthe scope of the present invention.

1. A pharmaceutical composition comprising a lipid nanoparticle and apharmaceutically acceptable excipient, wherein the lipid nanoparticlecomprises: a polynucleotide that is messenger RNA (mRNA); one or morenon-cationic lipids that are not a cholesterol-based lipid, one or morecholesterol-based lipids and/or one or more PEG-modified lipids; and oneor more chemical entities of formula I, each of which is a compound offormula I:

or a pharmaceutically acceptable salt thereof wherein 70% or more of thechemical entities of formula I have the structure set forth by formulaI.a.i,

2.-7. (canceled)
 8. The pharmaceutical composition of claim 1, wherein80% or more of the chemical entities of formula I have the structure setforth by formula I.a.i.
 9. The pharmaceutical composition of claim 1,wherein 90% or more of the chemical entities of formula I have thestructure set forth by formula I.a.i.
 10. The pharmaceutical compositionof claim 1, wherein 95% or more of the chemical entities of formula Ihave the structure set forth by formula I.a.i. 11.-47. (canceled)
 48. Amethod of delivery of messenger RNA (mRNA) in vivo, comprisingadministering to a subject in need of delivery the pharmaceuticalcomposition of claim 1, wherein administering of the lipid nanoparticleresults in the expression of the therapeutic protein encoded by the mRNAin vivo. 49.-60. (canceled)
 61. A method of treating a disease ordisorder comprising the step of delivering an mRNA encoding atherapeutic protein using the pharmaceutical composition of claim
 1. 62.The pharmaceutical composition of claim 1, wherein the mRNA isunmodified.
 63. The pharmaceutical composition of claim 1, wherein themRNA comprises one or more modified nucleotides.
 64. The pharmaceuticalcomposition of claim 63, wherein the one or more modified nucleotidescomprise pseudouridine, N-1-methyl-pseudouridine, 2-aminoadenosine,2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine,5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine,C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine,C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine,7-deaza-guanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine,and/or 2-thiocytidine.
 65. The pharmaceutical composition of claim 1,wherein the mRNA encodes a therapeutic protein.
 66. The pharmaceuticalcomposition of claim 1, wherein the pharmaceutical formulation is fororal, rectal, vaginal, transmucosal, pulmonary, or parenteraladministration.
 67. The pharmaceutical composition of claim 66, whereinthe pulmonary administration is intratracheal or intestinaladministration.
 68. The pharmaceutical composition of claim 66, whereinthe parenteral administration is intradermal, transdermal,intramuscular, subcutaneous, intramedullary, intrathecal, directintraventricular, intravenous, intraperitoneal, or intranasaladministration.
 69. The pharmaceutical composition of claim 65, whereinthe one or more non-cationic lipids are selected from the groupconsisting of DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC(1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE(1,2-dioleyl-sn-glycero-3-phosphoethanolamine), DOPC(1,2-dioleyl-sn-glycero-3-phosphotidylcholine), DPPE(1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE(1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), and DOPG(1,2-dioleoyl-sn-glycero-3-phospho-(l′-rac-glycerol)).
 70. Thepharmaceutical composition of claim 65, wherein the one or morecholesterol-based lipids are cholesterol and/or PEGylated cholesterol.71. The pharmaceutical composition of claim 65, wherein the one or morePEG-modified lipids comprise a poly(ethylene) glycol chain of up to 5kDa covalently attached to a lipid with alkyl chain(s) of C6-C20 length.72. A method of delivery of messenger RNA (mRNA) in vivo, comprisingadministering to a subject in need of delivery the pharmaceuticalcomposition of claim 69, wherein administering of the lipid nanoparticleresults in the expression of the therapeutic protein encoded by the mRNAin vivo.
 73. A method of delivery of messenger RNA (mRNA) in vivo,comprising administering to a subject in need of delivery thepharmaceutical composition of claim 70, wherein administering of thelipid nanoparticle results in the expression of the therapeutic proteinencoded by the mRNA in vivo.
 74. A method of delivery of messenger RNA(mRNA) in vivo, comprising administering to a subject in need ofdelivery the pharmaceutical composition of claim 71, whereinadministering of the lipid nanoparticle results in the expression of thetherapeutic protein encoded by the mRNA in vivo.